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Sample records for adenovirus-mediated gene transfer

  1. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  2. Angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium

    Institute of Scientific and Technical Information of China (English)

    DONG Shu-qiang; ZHANG Bao-ren; MEI Ju; XU Zhi-yun; ZOU Liang-jian; HUANG Sheng-dong

    2002-01-01

    Objective: To study the angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium. Methods: Domestic pigs underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, Ad. VEGF-B, Ad. LacZ or PBS were administrated directly into the myocardium at 10 sites in the circumflex distribution (109 PFU or 100 μl) according to groups. Echocardiography and ex vivo coronary angiography were performed. The injection sites around myocardium were harvested and subjected to histological analysis and immunochemical staining. Results: Echocardiography assessment 4 weeks after vector administration demonstrated significant improvement of regional wall systolic function. Collateral vesseldevelopment assessed by angiography was also significantly greater in Ad. VEGF-B animals than that in control animals. Vascular density analysis revealed a mean of 43±5 neovessels per high-power field in Ad.VEGF-B group versus 19±4 and 17±6 in Ad.LacZ and PBS group. Conclusion:Direct intramyocardial administration of Ad.VEGF-B can induce focal angiogenesis and result in improvement in regional myocardial function, which may be useful in patients with ischemic heart disease who are not eligible for conventional therapies.

  3. Suppression of gastric cancer growth by adenovirus-mediated transfer of the PTEN gene

    Institute of Scientific and Technical Information of China (English)

    Ying Hang; Yong-Chen Zheng; Yan Cao; Qing-Shan Li; Yu-Jie Sui

    2005-01-01

    AIM: To investigate the tumor-suppressive effect of the phosphatase and tensin homologue deleted from chromosome (PTEN) in human gastric cancer cells th atwere wild type for PTEN.METHODS: Adenoviruses expressing PTEN or luciferase as a control were introduced into gastric cancer cells.The effect of exogenous PTEN gene on the growth and apoptosis of gastric cancer cells that are wtPTEN were examined in vitro and in vivo.RESULTS: Adenovirus-mediated transfer of PTEN (AdPTEN) suppressed cell growth and induced apoptosis significantly in gastric cancer cells (MGC-803, SGC-7901)carrying wtPTEN in comparison with that in normal gastric epithelial cells (GES-1) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase and mitogen-activated protein kinase and cell-cycle arrest at the G2/M phase but not at the G1 phase. Furthermore,treatment of human gastric tumor xenografts (MGC-803,SGC-7901) with Ad-PTEN resulted in a significant (P<0.01)suppression of tumor growth.CONCLUSION: These results indicate a significant tumorsuppressive effect of Ad-PTEN against human gastric cancer cells. Thus, Ad-PTEN may be used as a potential therapeutic strategy for treatment of gastric cancers.

  4. Utility of adenovirus-mediated Fas ligand and bcl-2 gene transfer to modulate rat liver allograft survival

    Institute of Scientific and Technical Information of China (English)

    De-Sheng Wang; Yu Li; Ke-Feng Dou; Kai-Zong Li; Zhen-Shun Song

    2006-01-01

    BACKGROUND: Expression of Fas ligand (FasL) on the graft by gene transduction is expected to introduce apoptosis to lymphocytes to protect rejection, but the FasL-expressing graft cells may also induce apoptosis as the graft usually expresses Fas antigens. In this study, a strong antiapoptotic gene, bcl-2, was cotransfected with the FasL gene in rat liver graft to protect against Fas-mediated cell death and to prolong recipient survival. METHODS: Orthotopic liver transplantation was done in a strain combination of DA to LEW rats. After donor vascular isolation, adenovirus-mediated FasL and bcl-2 genes were cotransfected in the liver graft. RESULTS: Intragraft expression of FasL mRNA was constitutively expressed after adenovirus-mediated transduction, although expression of FasL increased mildly in control grafts. Bcl-2 mRNA was highly expressed at 2 days after reperfusion. In contrast, lower expression of bcl-2 was observed in the control group. The average survival of the gene transferred allografts increased from (9.8+1.3) days to (18.5+8.7) days compared with the control group. CONCLUSION: Our results indicate that rat liver allografts can be protected against host immune responses by adenovirus-mediated FasL and bcl-2 transfection, and that bcl-2 expression prevents the graft from Fas-mediated apoptosis.

  5. SYNERGISTIC EFFICACY OF ADENOVIRUS-MEDIATED BCL-XS GENE TRANSFER AND TOPOTECAN IN OVARIAN CANCER CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To observe the synergistic efficacy between Adenovirus-mediated bcl-Xs(Adv-bcl-Xs) gene transfer and chemotherapy on ovarian cancer cell growth. Methods: NuTu-19 cells were infected by different titers of Adv-bcl-Xs and treated with topotecan in the meantime. Cell proliferation was measured 3 days later by MTT. Graphical representations and statistical analyses for their interaction in tumor cells were done. Results: The statistical result and Graphical representations of the statistical modeling showed synergy effect on cell growth inhibition (P<0.01). Conclusion: There were synergistic efficacies between Adv-bcl-Xs gene therapy and Topotecan in ovarian cancer cell growth.

  6. EFFECT OF ADENOVIRUS-MEDIATED p53 GENE TRANSFER ON APOPTOSIS AND RADIOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张珊文; 肖绍文; 吕有勇

    2003-01-01

    Objective: To evaluate the effect of adenovirus- mediated p53 gene (Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines. Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 protein expression was detected by immunohistochemistry assay and western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infected with Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flow cytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection at Adp53 at 100 MOI which caused high transfer rate of wild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53 status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

  7. Adenovirus-mediated transfer of RA538 gene and its antitumor effect

    Institute of Scientific and Technical Information of China (English)

    程金科; 林晨; 隗玥; 张雪艳; 邢嵘; 牟巨伟; 王秀琴; 吴旻

    1999-01-01

    The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of cmyc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.

  8. Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    哈小琴; 李元敏; 劳妙芬; 苑宾; 吴祖泽

    2003-01-01

    Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer. Methods A total of 12 female New Zealand rabbits were used in this study. Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear. Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique. After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad-HGF group 1, Ad-HGF group 2, Ad-HGF group 3, Ad-GFP (a reporter gene) group and the solvent group. Immediately after surgery, 6×107 pfu Ad-HGF, 6×108 pfu Ad-HGF, 6×109 pfu of Ad-HGF, 6×109 pfu of Ad-GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively. One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad-GFP (6×109 pfu) into its wounds. Ice slides of wounds from this animal were observed under fluorescence microscopy. Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad-HGF (6×109 pfu) into each of its wound. Immunohistochemistry was used for detection. Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus-mediated gene transfer. Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over-expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis. Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring.Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing.

  9. Treatment of chronical myocardial ischemia by adenovirus-mediated hypatocyte growth factor gene transfer in minipigs

    Institute of Scientific and Technical Information of China (English)

    YUAN Biao; ZHANG YouRong; ZHAO Zhong; WU DanLi; YUAN LiZhen; WU Bin; WANG LiSheng; HUANG Jun

    2008-01-01

    Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno-virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab-lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran-domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

  10. Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

  11. Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer

    Institute of Scientific and Technical Information of China (English)

    WANG Hai-jun; YU Chang-long; Kishi Hiroyuki; Motoki Kazumi; MAO Ze-bin; Muraguchi Atsushi

    2006-01-01

    (cartilage). The samples were examined by light microscopy and quantitatively evaluated. Results Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-α blockade has little effect on joint destruction.Conclusion The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficaciousthan blockade of either cytokine alone in treating OA.

  12. Adenovirus-mediated human β-nerve growth factor gene transfer has a protective effect on cochlear spiral ganglion after blast exposure

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study whether adenovirus-mediated human β-nerve growth factor (Ad-hNGFβ) gene has any protective effect on blast hearing impairment. Methods:Deafness was induced by blast exposure (172. 0 dB) in 30 healthy guinea pigs. On day 7 of blast exposure, Ad-hNGFβ was infused into the perilymphatic space of 20 animals as the study group (hNGFβ group), and artificial perilymph fluid (APF) was infused into the perilymphatic space of the other 10 animals as the control group. At weeks 1, 4 and 8 after blast exposure, the animals were sacrificed and the cochleae were removed for immunohis-tochemical and HE stainings. Results: Expression of Ad-hNGFβ protein was detected in each turn of the cochlea at the 1st week, with almost equal intensity in all turns. At the 4th week, the reactive intensity of the expression of Ad-hNGFβ protein decreased. At the 8th week, no expression was detectable. The results of HE staining showed that the amount of spiral ganglions in hNGFβ group was significantly greater than that of the control group at week 4 (F<0. 01). Conclusion: Ad-hNGFβ can be expressed at a high level and for a relatively long period in the blast impaired cochlea, suggesting that Ad-hNGFβ has a protective effect on cochlear spiral ganglion cells after blast exposure and the efficient gene transfer into cochlea had been achieved without toxicity.

  13. Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

    International Nuclear Information System (INIS)

    Therapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors. Adenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production. The inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time. The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers

  14. ADENOVIRUS-MEDIATED P53 GENE TRANSFER INCREASES THE THERMOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES (IN VITRO AND IN VIVO)

    Institute of Scientific and Technical Information of China (English)

    张珊文; 肖绍文; 吕有勇

    2003-01-01

    Objective: To investigate the effect of adenovirus- mediated p53 (Adp53) transfer on thermosensitivity of human gastric carcinoma cell lines (BGC823). Methods: Two human gastric carcinoma cell lines with different p53 status, BGC823-wtp53 cell (abbreviate W) bearing the wilt-type p53 and BGC823-mutp53 cell (abbreviate M) bearing the mutant p53, were cultured with DMEM medium and were infected with Adp53 at a viral multiplicity of infection of 100 (1:100MOI) for 48h before heating. Cell cycle redistribution and apoptosis of two human gastric carcinoma cell lines in 24h at 37℃ after heat treatment at 42℃ for 2h or 43℃ for 0.5h were analyzed by flow cytometry. Relative tumor volume growth curves were used in a nude mouse tumor model of the two cell lines following hyperthermia at 43℃ for 0.5h after 48h intratumoral injection of 1(108 pfu of Adp53 to evaluate thermoenhancemet effect in vivo. Results: In vitro study showed that both W and M cells infected with Adp53 and treated with heating had strong arrest in G2 (after heating at 42℃ for 2h, 34.0% of original population for W cells and 25.3% of original population for M cells) and produced obvious apoptotic response. The apoptosis rate showed 230% increased (for W cells) and 110% increase (for M cells) compared with heating only control. In vivo study showed that the growth of tumor of both W cells and M cells was significantly delayed by hyperthemia combining with Adp53 as compared to tumors receiving either treatment alone. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and thermo- sensitivity in vitro and tumor thermosensitivity in vivo independent of cellular intrinsic p53 status. These results support the combined used of p53 gene therapy with hyperthermia in clinical trials.

  15. Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transfer Driver by KDR Promoter in Treatment of Experimental Human HepatocelLular Carcinoma in Nude Mice

    Institute of Scientific and Technical Information of China (English)

    LI Bao-jin; ZHANG Chao; YI Yuan-xue; HAO Ying; LIU Xiao-ping; OU Qing-jia

    2007-01-01

    Objective: To investigate the therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transfer under the driving of KDR promoter (AdKDR-tk) in combination of ganciclovir (GCV) against human hepatocellular carcinoma in nude mice. Methods: HepG2 cell line was implanted subcutaneously into 32 nude mice, which were subsequently divided into 4 groups (n=8 each group): Ganciclovir group (Ⅰ), Ad group (Ⅱ), AdCMV-tk/GCV group (under the driving of CMV promoter) (Ⅲ) and AdKDR-tk/GCV group (Ⅳ). Then intratumoral injection of recombinant adenovirus or Ad was performed in all nude mice, and repeated 24 h later. For the following 10 d GCV was given at a dose of 100 mg/(kg·d), ip. All the treated animals were killed to evaluate the tumor weight and the histopathological changes and the microvessel density of tumors after the treatment was determined. Results: Compared with group Ⅰ, the tumor inhibitory rate was 12.3% in group Ⅲ and 24.5% in group Ⅳ; the inhibition rates were significantly different between group Ⅲ and Ⅳ (P<0.05). The mean MVDs in group Ⅰ, Ⅱ, Ⅲand Ⅳ were 37.4±8.6, 30.6±7.8, 27.6±7.1, and 10.7±4.1 (microvessels/mm2), respectively. Significant differences were found between group Ⅲ and Ⅱ (P<0.05), Ⅳ and Ⅱ (P<0.01), and Ⅳ and Ⅲ (P<0.01). Conclusion: Intratumoral injection of AdKDR-tk results in marked inhibition of HCC growth through inhibition angiogenesis in nude mice. It may be a new treatment approach for human HCC.

  16. Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

    Science.gov (United States)

    Saito, Y; Swanson, X; Mhashilkar, A M; Oida, Y; Schrock, R; Branch, C D; Chada, S; Zumstein, L; Ramesh, R

    2003-11-01

    The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers. PMID:14528320

  17. Adenovirus-mediated hypoxia-inducible factor-1 alpha gene transfer induces angiogenesis and neurogenesis following cerebral ischemia in rats

    Institute of Scientific and Technical Information of China (English)

    Wanfu Wu; Xiu Chen; Zhen Yu; Changlin Hu; Wenqin Cai

    2008-01-01

    BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) accumulates under conditions of hypoxia. HIF-1α target genes have pleiotropic effects on neurogenesis, neuroprotection and angiogenesis in the brain.OBJECTIVE: To investigate whether a recombinant adenovirus carrying HIF-1α can increase the expression of HIF-1α in vivo and thus promote angiogenesis and neurogenesis in a rat model of focal cerebral ischemia.DESIGN, TIME AND SETTING: The randomized, controlled experiment was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA from September 2006 to October 2007.MATERIALS: 68 healthy adult male Sprague-Dawley (SD) rats, weighing 230-250 g, were used. HIF-1α antibody was purchased from Wuhan Boster Company. Vascular endothelial growth factor (VEGF) antibody was purchased from Santa Cruz Biotech Company.METHODS: All 68 rats were induced with a transient middle cerebral artery occlusion (MCAO), according to the method of intra-luminal vascular occlusion. 54 rats, in which MCAO was successfully induced, were randomly divided into adenovirus (Ad) group and recombinant adenovirus with HIF-1αgene (Ad-HIF-1α) group (27 rats for each group). Rats were injected with 10 μL Ad (Ad group) or Ad-HIF-1α (Ad-HIF-1α group) into the lateral ventricle, 1 day after MCAO induction. MAIN OUTCOME MEASURES: Reverse transcription polymerase chain reaction was used to measure the expression of HIF-1α and of VEGF. Immunohistochemistry was used to detect the localization of HIF-1α, VEGF and factor Ⅷ in ischemic penumbra. Rat newborn nerve cells were labeled with 5-bromodeoxyuridine (BrdU) after ischemia. BrdU/neurofilament 200 (NF200) and BrdU/glial fibrillary acidic protein (GFAP) double labeled immunofluorescent histochemistry was used to identify the differentiation of newborn cells. Neurological function was evaluated using the modified neurological severity score (NSS).RESULTS: Compared with Ad, Ad-HIF-1αenhanced the expression of HIF-1

  18. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  19. Up-regulation of integrin β3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy

    International Nuclear Information System (INIS)

    Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P<0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin β3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin β3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin β3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy. (author)

  20. Adenovirus-mediated interteukin-13 gene therapy attenuates acute kidney allograft injury

    NARCIS (Netherlands)

    Sandovici, Maria; Deelmani, Leo E.; van Goor, Harry; Helfrich, Wijnand; de Zeeuw, Dick; Henning, Robert H.

    2007-01-01

    Background Kidney transplantation is possible by virtue of systemic immunosuppression, which is in turn accompanied by serious side effects. The search for novel therapeutic agents and strategies is ongoing. Here we investigate the effects of adenovirus-mediated gene therapy with interleukin (IL)-13

  1. Increase in muscarinic stimulation-induced Ca(2+) response by adenovirus-mediated Stim1-mKO1 gene transfer to rat submandibular acinar cells in vivo.

    Science.gov (United States)

    Morita, Takao; Nezu, Akihiro; Tojyo, Yosuke; Tanimura, Akihiko

    2013-10-01

    Adenoviruses have been used for gene transfer to salivary gland cells in vivo. Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca(2+) responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca(2+) response was notable upon weak muscarinic stimulation and was attributed to increased Ca(2+) release from internal stores and increased Ca(2+) entry. The basal Ca(2+) level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca(2+) concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP3) produced similar effects on the release of Ca(2+) from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo, and may be applied to increase salivary secretions in experimental animals and human in future.

  2. Effects of adenovirus mediated vascular endothelial growth factor gene transfer on reconstitution of hematopoiesis in post-bone marrow transplantation mice

    Institute of Scientific and Technical Information of China (English)

    ZHONG Zhao-dong; ZOU Ping; HU Xian-shi; YOU Yong; CHEN Zhi-chao; HUANG Shi-ang

    2005-01-01

    Background Bone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study, adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice.Methods Recombinant adenovirus (Ad)-enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity.Results Significant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866.67±97.13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups (P0.05]. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control [(62.3±4.0)%, P<0.05] at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT [(46.5±5.0)% and (55.1±4.5)%] exceeded those of other BMT groups (P<0.05, respectively).Conclusion VEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice.

  3. Long-term doxycycline-controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat model of Parkinson’s disease

    OpenAIRE

    Corti, Olga; Sánchez-Capelo, Amelia; Colin, Philippe; Hanoun, Naïma; Hamon, Michel; Mallet, Jacques

    1999-01-01

    Developments of technologies for delivery of foreign genes to the central nervous system are opening the field to promising treatments for human neurodegenerative diseases. Gene delivery vectors need to fulfill several criteria of efficacy and safety before being applied to humans. The ability to drive expression of a therapeutic gene in an adequate number of cells, to maintain long-term expression, and to allow exogenous control over the transgene product are essential requirements for clini...

  4. Adenovirus-mediates gene transfer of brain-derived neurotrophic factor for repairing sciatic nerve injury%重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤

    Institute of Scientific and Technical Information of China (English)

    李培建; 李兵仓

    2011-01-01

    BACKGROUND: How to accelerate injury repair and regeneration following peripheral nerve injury is the research focus. Gene therapy may be the possible treatment for this problem.OBJECTIVE: To observe the expression of the brain-derived neurotrophic factor (BDN F) gene after microinjected adenovirus-mediated gene transfer of BDNF (AxCA-BDNF) to the sciatic nerve for peripheral nerve regeneration.METHODS: Based on silicone tube graft as a support to bridge adult rat sciatic nerve gaps, Wistar rat were microinjected recombinant adenovirus vector of BDNF (AxCA-BDNF), BDNF and simple injection of virus buffer to the sciatic nerve respectively.With the methods of in situ hybridization and immunocytochemistry, the BDNF gene expression was certified, the number of the new nerve fibers and motoneurons in anterior horn of the spinal cord were calculated, and the myelin sheath thickness of the new nerve fibers was measu red at 3, 7, 14 days and 1 , 2, 4 months after operation.RESULTS AND CONCLUSION: Compared with the BDNF and control group, the expression of the BDNF gene in the proximal end, distal end and spinal cord (L3-6) of injured sciatic nerve were obviously higher than that of the BDNF and control groups (P < 0.01). The result of retrograde axonal transport of HRP tracer indicated the survival neurons, regenerated nerve fibers,thickness of myelin sheath, as well as the re-formation of nerve connection of the AxCA-BDNF group were superior to the control group(P < 0.01). The results demonstrated that exogenous BDNF gene and its express proteins were uptaken to the spinal cord motoneurons through retrograde axonal transport. Gene therapy for sciatic nerve injury of adult rats by adenovirus-mediated gene transfer of brain-derived neurotrophic factor in vivo not only promotes nerve regeneration but also protects the neurons in the spinal cord.%背景:如何促进周围神经损伤修复与再生一直是基础与临床研究的热点.基因治疗有可能成为今后

  5. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  6. Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

    Institute of Scientific and Technical Information of China (English)

    崔磊; 李发成; 张群; 钱云良; 关文祥

    2003-01-01

    Objective: To evaluate the effect of local application of vascular endothelial growth factor (VEGF) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm×2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF (AdCMV-VEGF group, n=10), 1 012 pfu recombinant β-galactosidase adenovirus (AdCMV-Gal group, n=10) and 1 ml saline (saline group, n=10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation. Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P<0.01) and the saline group (P<0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.Conclusions: Local application of adenovirus-mediated VEGF165 cDNA 05- efficiently improve the survival of ischemic skin flaps.

  7. GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; HAN De-min; WANG Wen-ge; WU Zu-ze; ZHANG Wei

    1999-01-01

    Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer cell line Hep-2 was used.Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

  8. Adenovirus-mediated CTLA4Ig gene inhibits infiltration of immune cells and cell apoptosis in rats after liver transplantation

    Institute of Scientific and Technical Information of China (English)

    Guo-Ping Jiang; Zhen-Hua Hu; Shu-Sen Zheng; Chang-Ku Jia; Ai-Bin Zhang; Wei-Lin Wang

    2005-01-01

    AIM: To investigate the role of adenovirus-mediated CTLA4Ig gene therapy in inhibiting the infiltration of macrophages and CD8+T cells and cell apoptosis after liver transplantation.METHODS: The rat orthotopic liver transplantation model was applied. The rats were divided into three groups:group Ⅰ: rejection control (SD-to-Wistar); group Ⅱ: acute rejection treated with intramuscular injection of CsA injection of 1× 109 PFU adenovirus-mediated CTLA4Ig gene liquor in dorsal vein of penis 7 d before liver transplantation(SD-to-Wistar+CTLA4Ig). Immunohistochemistry and transferase-mediated dUTPnick-end labeling (TUNEL)were used to analyze the expression of CTLA4Ig gene in liver, infiltration of macrophages and CD8+T cells, cell apoptosis in grafts at different time-points after liver transplantation. Histopathological examination was done.RESULTS: CTLA4Ig gene expression was positive in liver on d 7 after administering adenovirus-mediated CTLA4Ig gene via vein, and remained positive until day 60 after liver transplantation. Infiltration of macrophages and CD8+T cells in CTLA4Ig-treated group was less than in rejection control group and CsA-treated group. The apoptotic index of rejection group on d 3, 5, and 7 were significantly higher than that of CTLA4Ig-treated group. A good correlation was found between severity of rejection reaction and infiltration of immune activator cells or cell apoptotic index in grafts.CONCLUSION: CTLA4Ig gene is constantly expressed in liver and plays an important role in inducing immune tolerance.

  9. 神经生长因子基因转染联合强化铁营养防治豚鼠爆震性聋的实验研究%Protective effects of adenovirus-mediated human bta-nerve growth factor gene transfer combined with iron fortified nutrition on blast hearing damage in guinea pigs

    Institute of Scientific and Technical Information of China (English)

    吴建; 武江; 范静平; 何金; 孙爱华

    2009-01-01

    目的 探讨人类神经生长因子β基因(human beta-nerve growth factor,hNGFβ)转染联合强化铁营养(fortified iron nutrition,FIN)防治豚鼠爆震性聋的可能性.方法 制作强脉冲噪声(172 dBSPL)致聋豚鼠模型35只,爆震后第7天,10只豚鼠经耳蜗底周鼓阶骨壁钻孔向外淋巴腔内导入腺病毒携带hNGFβ基因(adenovirus-mediated hNGFβ,Ad-hNGFβ)为基因组,10只豚鼠导入hNGFβ基因并进行强化铁营养为联合组,10只豚鼠经耳蜗底周鼓阶骨壁钻孔向外淋巴腔内导入人工外淋巴液(artificialperilymphatic fluid,APF)为APF组.5只豚鼠作正常对照组,不经暴露噪声,也不用药物治疗.测定爆震前及基因转染后豚鼠脑干听觉诱发电位(auditory brain stem response,ABR)阈值.取材时间:基因导入后第1周及第4周实验组各取5只动物进行耳蜗取材,并进行免疫组织化学染色和HE染色,检测Ad-hNGFβ蛋白表达并进行螺旋神经节细胞计数.结果 基因导入后第1周,可见Ad-hNGFβ在耳蜗内成功转染.耳蜗各回均有表达,强度基本相等;联合组豚鼠ABR反应阈恢复较基因组快,较APF组明显快;4周后,联合组豚鼠ABR反应阈完全恢复正常,基因组基本恢复正常,APF组未能恢复;联合组豚鼠螺旋神经节细胞数目多于基因组,两者均明显多于对照组,计数结果差异有统计学意义(P<0.01),且细胞形态与正常相近.结论 腺病毒介导的hNGFβ基因联合强化铁营养能协同作用防治豚鼠爆震性听力损伤.%Objective To study the protective effects of adenovirus-mediated human beta-nerve growth factor gene (hNGFβ) transfer combined with iron fortified nutrition on blast hearing damage in guinea pigs. Methods Deafness was induced by blast (172dB SPL) in 35 healthy guinea pigs. Seven days after noise exposure, 10 guinea pigs were inoculated with adenovirus-mediated hNGFβ (Ad-hNGFβ) into the perilymphatic space (the gene group), another 10 guinea pigs were given h

  10. p53基因转移至移植心脏的安全性%Security for adenovirus-mediated p53 gene transfer to the donor heart

    Institute of Scientific and Technical Information of China (English)

    王丽平; 宋芳芳; 李祥禄; 刘越; 贾智博; 尹新华

    2011-01-01

    BACKGROUND: Wild-typep53 gene transfer to the donor heart can greatry inhibit graft co to nan/ artery intima hyperplasia andlumen narrowness.OBJECT P/E: To study the security of adenoviral-mediated wild-type p53 gene transfer to the donor heart after hearttransplantation.METHODS: Rat modee of heterotopic (abdomen) heart transplantation over e developed. Wetar rats served as donors and SOrats as recipients. After donor hearts were removed. 800 u Ladenoviral vector encoding the wild-type p53gene(Ajdp53group)adenoviral vector encoding the &-galactosidase gene (LacZ) (Ad-LacZ group) or saline (control group) were infused into thedonor heart respectively before transplantation. The donor heart was stored in the 4 ~C saline for 30 minutes before hearttransplantation. At5 days after operation. P53 protein expressions in coronary artery of donor hearts were tested by western blotanalyse. £123 days after transplantation, the serum specimen was collected for the biochemical indicators, and the major organsof the recipients were tested by the hetopathological analysis and the reverse transcription polymerase chain reaction of theadenoviral E1A sequences.RESULTS AND CONCLUSION: The expression of P53 protein was found in donor hearts inAd-p53 group at 5 days afteroperation, and no expression in Ad-LacZ group and control group. At28 days after operation, rat serum biochemistry values inthree groups was normal, the major organs of the recipients were not affected seriously, no virus spread to other organs in theexperimental protocol. The results confirmed that the ex vivo adenoviral-mediated gene transfer to the donor heart via thecoronary artery during the heart transplantation e safe.%背景:课题组前期实验表明野生型p53基因具有抑制移植心脏冠状动脉内膜增厚的作用.目的:研究腺病毒介导的野生型p53基因转移至移植心脏的安全性.方法:以Wistar大鼠为供体,SD大鼠为受体建立大鼠腹腔异位心脏移植模型,在取出

  11. Reversal of 5-flouroucial resistance by adenovirus-mediated transfer of wild-type p53 gene in multidrug-resiatant human colon carcinoma LoVo/5-FU cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-Wei Yu; Peng Zhao; Ming Liu; Xin-Shu Dong; Ji Tao; Xue-Qin Yao; Xin-Hua Yin; Yu Li; Song-Bin Fu

    2004-01-01

    AIM: To observe the reversal effects of wide-type p53 gene on multi-drug resistance to 5-FU (LOVO/5-FU).METHODS: After treatment with Ad-p53, LOVO/5-FU sensitivity to 5-Fu was investigated using tetrazolium dye assay. Multidrug resistance gene-1 (MDR1) gene expression was assayed by semi-quantitative reverse transcriptionpolymerase chain reaction and the expression of p53 protein was examined by Western blotting.RESULTS: The reversal activity after treatment with widetype p53 gene was increased up to 4.982 fold at 48 h. The expression of MDR1 gene decreased significantly after treatment with wide-type p53 gene, and the expression of p53 protein lasted for about 5 d, with a peak at 48 h, and began to decrease at 72 h.CONCLUSION: Wide-type p53 gene has a remarkable reversal activity for the high expression of MDR1 gene in colorectal cancers. The reversal effects seem to be in a time dependent manner. It might have good prospects in clinical application.

  12. Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ya Ning; LI Qiang

    2014-01-01

    Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

  13. Adenovirus-mediated wild-type p53 gene transfer in combination with bronchial arterial infusion for treatment of advanced non-small-cell lung cancer, one year follow-up

    Institute of Scientific and Technical Information of China (English)

    Yong-song GUAN; Yuan LIU; Qing ZOU; Qing HE; Zi LA; Lin YANG; Ying HU

    2009-01-01

    Objective: In the present study, we have examined the safety and efficacy of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) injection in patients with advanced non-small-cell lung cancer (NSCLC) in the combination with the therapy of bronchial arterial infusion (BAI). Methods: A total of 58 patients with advanced NSCLC were enrolled in a non-randomized, two-armed clinical trial. Of which, 19 received a combination treatment of BAI and rAd-p53 (the combo group), while the remaining 39 were treated with only BAI (the control group). Patients were followed up for 12 months, with safety and local response evaluated by the National Cancer Institute's Common Toxicity Criteria and response evaluation criteria in solid tumor (RECIST), respectively. Time to progression (TTP) and survival rates were also analyzed by Kaplan-Meier method. Results: In the combo group,19 patients received a total of 49 injections of rAd-p53 and 46 times of BAI, respectively, while 39 patients in the control group received a total of 113 times of BAI. The combination treatment was found to have less adverse events such as anorexia, nausea and emesis, pain, and leucopenia (P0.05). Patients in the combo group had a longer TTP than those in the control group (a median 7.75 vs 5.5 months, P=0.018). However, the combination treatment did not lead to better survival, with survival rates at 3, 6, and 12 months in the combo group being 94.74%, 89.47%, and 52.63%, respectively, com-pared with 92.31%, 69.23%, and 38.83% in the control group (P=0.224). Conclusion: Our results show that the combination of rAd-p53 and BAI was well tolerated in patients with NSCLC and may have improved the quality of life and delayed the disease progression. A further study to better determine the efficacy of this combination therapy is warranted.

  14. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma.

    OpenAIRE

    M. CARUSO; Pham-Nguyen, K; Kwong, Y. L.; Xu, B; Kosai, K I; Finegold, M; Woo, S L; Chen, S. H.

    1996-01-01

    Recombinant adenoviral mediated delivery of suicide and cytokine genes has been investigated as a treatment for hepatic metastases of colon carcinoma in mice. Liver tumors were established by intrahepatic implantation of a poorly immunogenic colon carcinoma cell line (MCA-26), which is syngeneic in BALB/c mice. Intratumoral transfer of the herpes simplex virus type 1 thymidine kinase (HSV-tk) and the murine interleukin (mIL)-2 genes resulted in substantial hepatic tumor regression, induced an...

  15. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S;

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone...... or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  16. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  17. Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model.

    Science.gov (United States)

    Tsung, Andrew J; Kargiotis, Odysseas; Chetty, Chandramu; Lakka, Sajani S; Gujrati, Meena; Spomar, Daniel G; Dinh, Dzung H; Rao, Jasti S

    2008-03-01

    Matrix metalloproteinases (MMPs) comprise a class of secreted zinc-dependent endopeptidases implicated in the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2 (MMP-2) has been detected in high levels and correlates with invasiveness in human melanoma. We have studied the effect of adenovirus-mediated transfer of small interfering RNA (siRNA) against MMP-2 in the human melanoma cell line A2058. The delivery of these double-stranded RNA molecules represents an efficient technology in silencing disease-causing genes with known sequences at the post-transcriptional level. siRNA against MMP-2 mRNA (Ad-MMP-2) was found to decrease MMP-2 protein expression and activity in melanoma cells as demonstrated by western blotting and gelatin zymography. Furthermore, infection of cells with Ad-MMP-2 inhibited cellular migration and invasion as indicated by spheroid and matrigel assays. We also observed dose-dependent suppression of vascular network formation in an angiogenesis assay. Finally, we developed a nude mouse spinal metastatic model to investigate the local effects of tumor metastasis. Intravenous tail vein injection with Ad-MMP-2 on days 5, 9 and 11 after tumor implantation resulted in complete retention of neurological function as compared to control and scrambled vector (Ad-SV)-treated groups that showed complete paraplegia by day 14+/-2 days. Hematoxylin and eosin staining revealed decreased tumor size in the Ad-MMP-2-treated animals. This novel experimental model revealed that adenoviral-mediated transfer of RNA interference against MMP-2 results in the retention of neurological function and significantly inhibited tumor growth.

  18. Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system

    Science.gov (United States)

    Vasquez, E. C.; Beltz, T. G.; Haskell, R. E.; Johnson, R. F.; Meyrelles, S. S.; Davidson, B. L.; Johnson, A. K.

    2001-01-01

    The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin. Copyright 2000 Academic Press.

  19. Adenovirus-mediated human brain-derived neurotrophic factor gene-modified bone marrow mesenchymal stem cell transplantation for spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Changsheng Wang; Jianhua Lin; Chaoyang Wu; Rongsheng Chen

    2011-01-01

    Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1, 3, and 5 weeks after transplantation, the expression of ??brain-derived neurotrophic factor and neurofilament-200 was upregulated in the injured spinal cord, spinal cord injury was alleviated, and Basso-Beattie-Bresnahan scores of hindlimb motor function were significantly increased. This evidence suggested that intravenous transplantation of adenovirus- mediated brain-derived neurotrophic factor gene-modified rat bone marrow mesenchymal stem cells could play a dual role, simultaneously providing neural stem cells and neurotrophic factors.

  20. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S;

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in c...

  1. Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Qi; CHEN Dayu; FU Xiangning; ZU Yukun

    2006-01-01

    The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000 μg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

  2. Expression of adenovirus-mediated neurotrophin-3 gene in Schwann cells of sciatic nerve in rats

    Institute of Scientific and Technical Information of China (English)

    朱锦宇; 黄耀添; 朱庆生; 吕荣

    2003-01-01

    Objective: To investigate the expression of neurotrophin-3 (NT-3) gene in Schwann cells of rat sciatic nerve introduced by an adenovirus vector in vivo. Methods: A recombinant adenovirus vector for NT-3 (Ad-NT-3) was propagated in 293 packaging cells and titered with tissue culture infectious dose50 (TCID50). Ad-NT-3 was injected directly into the rat sciatic nerve after transection and immediate repair. Immunohistochemical staining was employed to determine the expression of NT-3 in Schwann cells in rat sciatic nerve and the expressive intensity of the tissue slices of the sciatic nerve was measured with LEICA M550 image analysis system. Results: On the 2nd day after injection of Ad-NT-3, positive stain in the Schwann cells was apparent in the vicinity of anastomosis. NT-3 expression increased significantly on the 7th day (P0.05). Compared with the 2nd day group, the 14th and 28th day groups still maintained a relatively high level of NT-3 (P<0.01). Intact and repaired nerves, which were injected with adenovirus encoding LacZ genes (Ad-LacZ) or physiological saline served as controls, showed no NT-3-positive Schwann cells. Conclusions: An adenovirus vector can be used to induce efficiently the expression of NT-3 gene in Schwann cells of rat peripheral nerves following nerve injury and repair, which suggests that neurotrophic factors can be introduced into Schwann cells with an adenovirus vector to promote peripheral nerve regeneration.

  3. Infection with adenovirus-mediated luciferase reporter gene in mesenchymal stem cells and bioluminescence imaging

    International Nuclear Information System (INIS)

    Objective: To construct adenovirus vector containing firefly luciferase reporter gene (Ad-Luc) and infect bone marrow mesenchymal stem cells (BMSC), then to take bioluminescence imaging in vitro and in vivo for identification. Methods: The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV). It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing. PShuttle-CMV-Luc and backbone vector (pAdeno) were homologous recombined. Then the recombinant plasmid was packaged in HEK293 cells and the virus titer was detected. The BMSC were infected by the recombinant adenovirus. The bioluminescence imaging in vitro was performed to determine the best multiplicity of infection (MOI), and the relationship between bioluminescence intensity and MOI was analyzed by curve fitting regression analysis. Viability was evaluated via Trypan blue staining. The transfected BMSC (1 × 106) were implanted into the muscles of forelimb of SD rats,and then tracked by bioluminescence imaging in vivo. Cell viability was compared using two-way repeated measures analysis of variance between groups. Results: Enzyme digestion and sequence analysis indicated that Ad-Luc was successfully constructed. The virus titer was 1 × 1010 plaque forming unit (PFU)/ml. The bioluminescence detection in vitro showed that Ad-Luc could infect BMSC high efficiently to express luciferase and the best MOI was 50. The bioluminescence intensity enhanced with increase of MOI (R2 =0.98). No statistically significant difference was found in cell viability between transfected and untransfected BMSC at 1, 3, 5, 7 d. The cell survival rates were (92.5±2.3)% vs (94.1±1.8)%, (91.4±0.9)% vs (92.7±2.0)%, (92.1±1.6)% vs (93.3± 2.4)%, (91.9 ± 1.5)% vs (93.0 ± 3.1)%, respectively (F=4.38, P>0.05). The bioluminescence imaging in vivo showed that BMSC survived 1, 3, 7 d after implantation. However, bioluminescence signal decreased gradually over time

  4. Adenovirus-mediated expression of human sodium-iodide symporter gene permits in vivo tracking of adipose tissue-derived stem cells in a canine myocardial infarction model

    International Nuclear Information System (INIS)

    Introduction: In vivo tracking of the transplanted stem cells is important in pre-clinical research of stem cell therapy for myocardial infarction. We examined the feasibility of adenovirus-mediated sodium iodide symporter (NIS) gene to cell tracking imaging of transplanted stem cells in a canine infarcted myocardium by clinical single photon emission computed tomography (SPECT). Methods: Beagle dogs were injected intramyocardially with NIS-expressing adenovirus-transfected canine stem cells (Ad-hNIS-canine ADSCs) a week after myocardial infarction (MI) development. 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) and 99mTc-pertechnetate (99mTcO4−) SPECT imaging were performed for assessment of infarcted myocardium and viable stem cell tracking. Transthoracic echocardiography was performed to monitor any functional cardiac changes. Results: Left ventricular ejection fraction (LVEF) was decreased after LAD ligation. There was no significant difference in EF between the groups with the stem cell or saline injection. 125I uptake was higher in Ad-hNIS-canine ADSCs than in non-transfected ADSCs. Cell proliferation and differentiation were not affected by hNIS-carrying adenovirus transfection. 99mTc-MIBI myocardial SPECT imaging showed decreased radiotracer uptake in the infarcted apex and mid-anterolateral regions. Ad-hNIS-canine ADSCs were identified as a region of focally increased 99mTcO4− uptake at the lateral wall and around the apex of the left ventricle, peaked at 2 days and was observed until day 9. Conclusions: Combination of adenovirus-mediated NIS gene transfection and clinical nuclear imaging modalities enables to trace the fate of transplanted stem cells in infarcted myocardium for translational in vivo cell tracking study for prolonged duration

  5. Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model

    International Nuclear Information System (INIS)

    Purpose: The purpose of this study was to evaluate the efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in an adjuvant setting with external beam radiation therapy (EBRT) in an experimental prostate cancer model in preparation for a Phase I clinical study in humans. Methods: For efficacy studies, i.m. DU145 and intraprostatic LNCaP C4-2 tumors were established in immune-deficient mice. Tumors were injected with the lytic, replication-competent Ad5-CD/TKrep adenovirus containing a cytosine deaminase (CD)/herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene. Two days later, mice were administered 1 week of 5-fluorocytosine + ganciclovir (GCV) prodrug therapy and fractionated doses of EBRT (trimodal therapy). Tumor control rate of trimodal therapy was compared to that of EBRT alone. For toxicology studies, immune-competent male mice received a single intraprostatic injection (1010 vp) of the replication-competent Ad5-CD/TKrep adenovirus. Two days later, mice were administered 4 weeks of 5-fluorocytosine + GCV prodrug therapy and 56 Gy EBRT to the pelvic region. The toxicity of trimodal therapy was assessed by histopathologic analysis of major organs and clinical chemistries. Results: In both the i.m. DU145 and intraprostatic LNCaP C4-2 tumor models, trimodal therapy significantly improved primary tumor control beyond that of EBRT alone. In the DU145 model, trimodal therapy resulted in a tumor growth delay (70 days) that was more than twice that (32 days) of EBRT alone. Whereas EBRT failed to eradicate DU145 tumors, trimodal therapy resulted in 25% tumor cure. In the LNCaP C4-2 tumor model, EBRT slowed the growth of intraprostatic tumors, but resulted in no tumor cures, and 57% of the mice developed retroperitoneal lymph node metastases at 3 months. By contrast, trimodal therapy resulted in 44% tumor cure and reduced significantly the percentage (13%) of lymph node metastases relative to EBRT alone. Overall

  6. 腺病毒介导的白介素-24转移对脂多糖诱导的大鼠肾小球系膜细胞凋亡和周期调节蛋白p21、p27及CyclinE的影响%Effects of adenovirus mediated IL-24 gene transfer on apoptosis and cell cycle regulatory protein p21,p27 and CyclinE of rat gomerular mesangial cells induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    王晓浪; 周建华; 王从俊

    2014-01-01

    Objective To explore the effect of interleukin-24(IL-24)gene transfer on glomerular mesangial cells(GMCs) apoptosis and to find out the effect of IL-24 on cell cycle regulatory protein p21,p27 and CyclinE of GMCs induced by LpS. Methods 293 cells were cultured in 10%FBS/DMEM and Ad. IL-24 and Ad. GFp were amplifycated in 293 cells. GMCs were analysed after 4 to 6 generations. ①They were divided into four groups:control group,Ad. IL-24 group,LpS group and LpS+Ad. IL-24 group. And control group and LpS group werenˊt infected with Ad. IL-24,Ad. IL-24 group and LpS+Ad. IL-24 group GMCs were infected with Ad. IL-24,then LpS+Ad. IL-24 group GMCs were cultured in 5%FBS/DMEM with LpS(10 mg·L-1 ). The apoptosis of the GMCs was examined by AnnexinV/FITC flow cytometry;②The effect of IL-24 on cell cycle regulatory protein p21, p27 and CyclinE of GMCs induced by LpS were determined. They were divided into three groups:control group,Ad-GFp group and IL-24 group. Control group GMCs were cultured in 5%FBS/DMEM. Ad-GFp group GMCs were infected with Ad. GFp and then cultured in 5%FBS/DMEM with LpS(10 mg·L-1 ). GMCs were infected with Ad. IL-24. The expressions of cell cycle regulatory protein p21,p27 and cyclinE were examined by Western-blotting. Results The GMCs were cultured for 24 hours and 48 hours. The apoptosis rate was(0. 86 ± 0. 15)% and(0. 98 ± 0. 4)% in the control group,(1. 02 ± 0. 22)% and(1. 43 ± 0. 31)% in the Ad. IL-24 group,(2. 19 ± 0. 81)% and(2. 49 ± 0. 12)% in the LpS group,(18. 01 ± 1. 17)% and(26. 82 ± 5. 01)% in LpS + Ad. IL-24 group. There was no difference between control group and Ad. IL-24 group,and the apoptosis rate of LpS group was higher than control group(P<0. 05). The apoptosis rate of LpS+Ad. IL-24 group was the highest while there was no change in Ad. IL-24 group(P<0. 05). ②The expressions of p21 and p27 were down-regulated while CyclinE expression was up-regulated in GMC by LpS(P<0. 05). Adenovirus mediated IL-24 gene transfer

  7. Oncolytic adenovirus-mediated transfer of the antisense chk2 selectively inhibits tumor growth in vitro and in vivo.

    Science.gov (United States)

    Chen, G; Zhou, J; Gao, Q; Huang, X; Li, K; Zhuang, L; Huang, M; Xu, G; Wang, S; Lu, Y; Ma, D

    2006-10-01

    Screening and identifying molecules target to checkpoint pathways has fostered the development of checkpoint-based anticancer strategies. Among these targets, inhibition of chk2 may induce cell death for tumors whose growth depends on enhanced chk2 activity. However, improvement of the potency and specificity of such therapeutics remains a major challenge. To resolve this problem, we constructed M3, a novel recombinant adenovirus with a 27-bp deletion in E1A CR2 region by which to realize tumor-specific replication, and an 829-bp of antisense chk2 fragment inserted into the E3 coding region. In this design, M3 exploited the native adenovirus E3 promoters to express antisense chk2 cDNA in a viral replication-dependent fashion, and preferentially silenced the chk2 gene in tumor cells. In vitro and in vivo assays confirmed that downregulated chk2 expression induced by M3 infection was tumor-specific and virus replication-dependent. Furthermore, systemic administration of M3 combined with a low dose of cisplatin cured 75% (9/12) of orthotopic hepatic carcinoma mouse models that were otherwise resistant to cisplatin. Our results indicated that the upcoming development in this field would improve the antitumor efficacy and maximize the synergistic effect of oncolytic viruses administered with traditional chemotherapy or radiotherapy. PMID:16741520

  8. 组织因子途径抑制物基因转移对大鼠血管平滑肌细胞凋亡的影响%Effect of adenovirus-mediated tissue factor pathway inhibitor gene transfer on apoptosis of rat vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    傅羽; 张改改; 曹滢; 刘越; 于金凤; 尹新华

    2009-01-01

    目的 研究组织因子途径抑制物(TFPI)基因转移对大鼠血管平滑肌细胞(VSMC)凋亡的影响,探讨其抑制血管成形术后再狭窄的机制.方法 在体外培养的大鼠VSMC中分别转染含有人TFPI基因的重组腺病毒(Ad-TFPI)、含β-半乳糖苷酶基因的重组腺病毒(Ad-LacZ)或PBS.通过RT-PCR方法检测外源TFPI基因的表达.细胞计数和MTT法测定细胞生长情况.电镜技术、流式细胞仪和TUNEL法分别检测细胞凋亡情况.结果 基因转移后3 d在VSMC中检测到TFPI mRNA的表达.细胞计数结果显示第1、3、5天各组细胞数无明显差异,而第7天Ad-TFPI组细胞数明显少于Ad-LacZ组和PBS组(P<0.05).MTT结果显示基因转移后第1、3、5天各组细胞的吸光度值比较差异无统计学意义,而第7天Ad-TFPI组的吸光度值明显低于Ad-LacZ组(P<0.05)和PBS组(P<0.01).流式细胞仪检测结果显示基因转移后3、5、7 d Ad-TFPI组细胞早期凋亡结果均高于Ad-LacZ组.基因转移后3 d和7 d,Ad-TFPI组TUNEL阳性率分别为(10.82±1.57)%和(16.95±2.01)%,明显高于Ad-LacZ组(3.46±0.93)%和(5.11±1.29)%(P<0.05).透射电镜结果显示基因转移后3、5、7 d Ad-TFPI组细胞逐渐出现体积变小、线粒体轻度肿胀、核固缩以及凋亡小体形成,而Ad-LacZ组则无明显改变.结论 TFPI基因转移能够显著诱导体外培养的大鼠VSMC发生凋亡,可能是其抑制血管成形术后再狭窄的机制之一.%Objective Previous studies showed potential role of tissue factor pathway inhibitor (TFPI)on attenuating restenosis,we investigated the effect of TFPI gene transfer on vascular smooth muscle cells(VSMCs)apoptosis.Methods Human TFPI recombinant adenovims or LacZ recombinant adenovirus or PBS were transferred t0 rat aortic VSMCs respectively in vitro.RT-PCR was used to detect the expression of exogenous TFPI gene.VSMCs were examined by cell counting and MTT.Apoptosis of VSMCs was detected by flow cytometry.TUNEL and

  9. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    International Nuclear Information System (INIS)

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer

  10. Prospective Randomized Phase 2 Trial of Intensity Modulated Radiation Therapy With or Without Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy in Intermediate-Risk Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Freytag, Svend O., E-mail: sfreyta1@hfhs.org [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Stricker, Hans [Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan (United States); Lu, Mei [Public Health Sciences, Henry Ford Health System, Detroit, Michigan (United States); Elshaikh, Mohamed; Aref, Ibrahim; Pradhan, Deepak; Levin, Kenneth; Kim, Jae Ho [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Peabody, James [Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan (United States); Siddiqui, Farzan; Barton, Kenneth; Pegg, Jan; Zhang, Yingshu; Cheng, Jingfang [Department of Radiation Oncology, Henry Ford Health System, Detroit, Michigan (United States); Oja-Tebbe, Nancy; Bourgeois, Renee [Public Health Sciences, Henry Ford Health System, Detroit, Michigan (United States); Gupta, Nilesh; Lane, Zhaoli [Pathology, Henry Ford Health System, Detroit, Michigan (United States); Rodriguez, Ron [Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); DeWeese, Theodore [Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); and others

    2014-06-01

    Purpose: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. Methods and Materials: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. Results: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. Conclusions: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

  11. Adenovirus-mediated REIC/Dkk-3 gene therapy: Development of an autologous cancer vaccination therapy (Review)

    OpenAIRE

    Watanabe, Masami; Nasu,Yasutomo; Kumon, Hiromi

    2013-01-01

    Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor suppressor and therapeutic gene and has been studied with respect to the application of cancer gene therapy. Our previous studies demonstrated that the intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) suppresses tumor growth in mouse models of prostate, breast and testicular cancer and malignant mesothelioma. The mechanisms underlying these antitumor therapeutic effects have ...

  12. Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

    Institute of Scientific and Technical Information of China (English)

    Mingyue Xiong; Kunzheng Wang

    2009-01-01

    Objective:To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine(MK) gene in pancreatic cancer cells. Methods:Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1a, 3rd, 5th, 7th, and 9th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results:The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3rd, 5th, and 7th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion:Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

  13. Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension

    OpenAIRE

    Morecroft, Ian; White, Katie; Caruso, Paola; Nilsen, Margaret; Loughlin, Lynn; Alba, Raul; Reynolds, Paul N; Danilov, Sergei M.; Andrew H. Baker; MacLean, Margaret R.

    2012-01-01

    Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmo...

  14. Preliminary study of MR diffusion weighted imaging in nude mice models of hepatic Bel7402 tumors after adenovirus-mediated cytosine diaminase-thymidine kinase gene therapy

    International Nuclear Information System (INIS)

    Objective: To study the characteristics of DWI in nude mice models of hepatic Bel7402 tumors after treatment with adenovirus-mediated cytosine diaminase-thymidine kinase (Ad. CD-TK) double suicide gene therapy, and then to identify whether DWI can be used for assessing curative effect of postoperative tumors. Methods: Thirty nude mice models of hepatic Bel7402 tumors were successfully created using cell suspension method, after the tumor grew to more than 1 cm in diameter, 20 tumor models were treated by intratumoral administration of Ad. CD-TK for 3 days plus intraperitonea (i.p.) treatment with 5-Fc and GCV for the duration of the study.Then they were randomly divided into three groups during 5-Fc and GCV treatment. The remaining 10 tumor models were used as controls. MR scanning were performed in 10th day before and after tumor implantation in all models by using EPI-SE series and SENSE technology for treatment group. Tumor volumes and ADC values were calculated pretreatment and posttreatment. Cell apoptosis were determined by using TUNEL method. Analyze the change of ADC and apoptosis index (AI) in different times, t test was used for comparison the difference of AI and ADC values respectively. Results: After 10 days,the tumor volumes of the treatment groups and controls were respectively (724.16 ±57.45) mm3, (754.57 ± 66.84) mm3, with no significant difference (t=0.488, P >0.05). The ADC values of the treatment groups were (0.98 ±0.11) × 10-3 mm2/s,the ones of the control groups were (0.68 ±0.04) × 10-3 mm2/s; AI of the treatment groups were (23.25 ±6.57)%, the ones of the control groups were (2.57 ± 0.58)%. There were difference in both groups (t=4.473, 5.874; P<0.01). Conclusion: DWI can be effectively to monitor the early pathological changes of hepatic Bel7402 tumors after Ad. CD-TK double suicide gene therapy, and provide experimental evidences for clinical application. (authors)

  15. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    International Nuclear Information System (INIS)

    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer

  16. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Haixi [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Li, Lili [Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute (Hong Kong); Ren, Guosheng [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Xu, Yongzhu [Chongqing Health Service Center, Chongqing 400020 (China); Zhou, Xiangyang [The Wistar Institute, Philadelphia, PA (United States); Xiang, Tingxiu, E-mail: xiangtx1@gmail.com [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China)

    2015-03-15

    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

  17. Adenovirus-mediated HSV-TK Gene Therapy Using hTERT Promoter in CNE Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu; YU Xiang-hui; ZHA Xiao; KONG Wei

    2009-01-01

    Human telomerase reverse transcriptase(hTERT) activity was detected in human nasopharyngeal carci-noma ceII(CNE) but not in human normal lung fibroblas t(CCD-11Lu). Recombinant adenoviruses Ad-CMV-TK-enh and Ad-hTERT-TK-enh were constructed and infected into normal fibroblasts and nasopharyngeal carcinoma cells. Ad-CMV-TK-enh with 100 μmol/L of ganciclovir(GCV) caused 87% of CCD-11 Lu cells death and 91% of CNE cells death, Ad-hTERT-TK-enh with 100 μmol/L of GCV caused 24% of CCD-11Lu cells death and 79% of CNE cells death. These results indicate that the Ad-hTERT-TK-enh with GCV may be a useful method in suppressing tumor growth in targeted nasopharyngeal carcinoma gene therapy.

  18. BCL-XS adenovirus-mediated gene therapy approach sensitizes cancer cells to radiation-induced apoptosis

    International Nuclear Information System (INIS)

    Purpose: Apoptosis, a process in which a genetic program is activated ultimately leading to programmed cell death, has been shown to play a role in radiation therapy (RT)-induced cell death. We and others have previously shown that members of the bcl-2 family (including bcl-xl) protect cells from RT-induced apoptosis through p53-dependent and -independent pathways. Therefore, we postulated that inactivation of bcl-2 family members by overexpression of bcl-xs (a functional inhibitor of the bcl-2 family) would enhance RT-induced apoptosis. Overexpression of bcl-xs was achieved using two strategies: stable transfection and transient infection using an adenovirus (AV) vector. Methods: An expression plasmid encodingbcl-xs (pSFFVneo-bcl-xs) or a control plasmid (pSFFVneo) was stably transfected into MCF-7 (breast cancer), K562 (human leukemia), and FL512 (pro B-cell) cells and clonogenic survival was determined following RT. The second method used to overexpress bcl-xs involved construction of an AV vector that expresses bcl-xs by inserting the bcl-xs coding sequence into the pADRSV vector. Immunoblotting using a rabbit polyclonal antibody raised against the bcl-x protein revealed that K562 cells infected with the bcl-xs AV, but not the control AV that contains the β-galactosidase gene, expressed the 21 kDA bcl-xs protein. K562 cells were infected with the bcl-xs AV or the control AV at titres to achieve 90-95% infection. Various doses of RT were given 24 hrs following infection since maximal expression of bcl-xs was achieved at this time. Colony forming ability following RT was performed. Apoptotic death at 24 and 48 hrs following RT was assayed by flow cytometry using propidium iodide which quantitates DNA damage. Results: Bcl-xs overexpression by stable transfection in all three cell lines tested induced a marked increase in radiosensitivity. Bcl-xs overexpressing K562, FL512, and MCF-7 cells were more sensitive to RT-induced clonogenic death than their neo

  19. [Adenovirus-mediated delivery of nm23-H1 gene inhibits growth of colorectal carcinoma cell line Lovo].

    Science.gov (United States)

    Wang, Qi; He, Xueling; Liu, Yan; Yin, Hailin

    2010-12-01

    This experimental study sought to find out the inhibitory effects of Ad-GFP-nm23-H1 on proliferation and metastasis of human colorectal carcinoma cell line Lovo, and, further, to gain an insight into some theoretical and methodical basis for instituting nm23-H1 gene therapy of cancers. MTT assay and Transwell chamber were used to detect the rates of proliferation and invasion as well as the adhesion of Lovo cells in vitro. The results demonstrated that the proliferation inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 84.9% +/- 1.51%, 48.5% +/- 7.23% and 22.5% +/- 5.47%, that the adherence inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 70.3% +/- 2.40%, 60.1% +/- 5.68% and 18.5% +/- 3.61%, and that the invasiveness inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 83.2% +/- 5.71%, 52.2% +/- 6.94% and 28.1% +/- 8.21%. These data suggested that Ad-GFP-nm23-H1 exerted significant inhibitory effects on the proliferation and metastasis of human colorectal carcinoma cell line Lovo in a dose-dependent way.

  20. 腺病毒介导的hCTLA4-Ig和FasL基因转移诱导大鼠同种异体肾移植长期存活的作用%Adenovirus-mediated CTLA4-Ig and FasL gene transfer induces long-term survival of renal allografts in rats

    Institute of Scientific and Technical Information of China (English)

    平季根; 温端改; 侯建全; 吕金星; 严春寅

    2009-01-01

    Objective To investigate the potential role of adenovirus-CTLA4-Ig and adenovirus-FasL recombinant in inducing transplantation tolerance using renal-graft model and its related mecha-nisms. Methods Allogeneic kidney transplants were performed between SD donors and Wistar recipients. The experimental rats were divided into 4 groups. In Ad-CTLA4-Ig group and Ad-CTLA4-Ig + Ad-FasL group, the donor kidney of the SD rats was locally transfected by Ad-CTLA4-Ig and Ad-CTLA4-Ig + Ad-FasL with the dose of 1 × 10~9-5 × 10~9 PFUml respectively and then transplanted to the recipient Wistar rats. In control group, the kidneys of the SD rats were directly transplanted to Wistar rats without any thera-py. The rats treated with Ad-EGFP served as empty vector group. After kidney transplantation, the survival time and the kidney function in each group were observed. Kidney allografts were evaluated by HE staining and immunohistochemical staining. The pathological features and ultrastructures of the grafts were ob-served. Results The survival time of allografts were prolonged significantly in recipients receiving Ad-CT-LA4-Ig + Ad-FasL with a mean survival time of (64.67 ± 6.41) days ,significantly longer than that in Ad-CTLA4-Ig treated group (31.33±6.77) days,control group (8.17 ± 1.17) days and empty vector group (8.00 ± 1.55) days (P < 0.01). After transplantation, the levels of creatinine in serum were significantly higher in control group and empety vector group than in Ad-CTLA4-Ig + Ad-FasL treated group and Ad-CTLA4-Ig treated group. Conclusion Adenoviral vectors can be successfully transduced into rat kidneys with the CTLA4-Ig and FasL cDNA. Ad-mediated transduction of the CTLA4-Ig and FasL gene can signifi-cantly prolong the survival of rat renal allograft. The induced tolerance is donor specific, and may result from regulatory T cells and the deletion of alloreactive T cells.%目的 探讨腺病毒介导hCTLA4-Ig和FasL基因转移延长异基因大鼠肾移植物

  1. Adenovirus-mediated transfer of VEGF into marrow stromal cells combined with PLGA/TCP scaffold increases vascularization and promotes bone repair in vivo

    OpenAIRE

    Duan, Chunguang; Liu, Jian; YUAN, ZHI; Meng, Guolin; Yang, Xiumei; Jia, Shuaijun; Zhang, Jinkang; Chen, Shi

    2012-01-01

    Introduction Large osseous defect remains a serious clinical problem due to the lack of sufficient blood supply and it has been proposed that this situation can be relieved by accelerating the formation of new vessels in the process of bone defect repair. The aim of this study was to develop a new type of artificial bone by transferring the VEGF gene into marrow stromal cells (MSCs) and seeding them into a porous scaffold. Material and methods An adenovirus vector was employed to transfer the...

  2. In vivo study on the effect of adenovirus mediating Smad 7 gene expression regulated by radiation via Egr-1 promoter in C57BL mice implanted with lewis lung cancer

    International Nuclear Information System (INIS)

    Objective: Objective To study the effect of adenovirus mediating Smad 7 gene regulated by radiation via Egr-1 on the primary tumor and lung metastasis in C57BL mice implanted with Lewis lung cancer. Methods: The radio-inducible elements from the Egr-1 gene promoter were inserted upstream to a cDNA encoding Smad 7 and integrated into a replication-defective adenovirus to generate recombinant adenovirus (AD. Egr-Smad 7). 270 mice implanted with Lewis lung cancer in the hind legs were used and the experiment was started when the transplanted tumor diameter reached 0.8 to l.0 cm. Then three investigations were undertaken, each demanding 90 mice implanted with Lewis lung cancer respectively. To each group, 90 mice models were randomized into 3 groups: the normal control group; the NS control group; and the implanted AD. Egr-Smad 7 group. Every 6 mice in each group were irradiated by different single close to study the following: 1. The maximal and minimal diameters of the tumor were recorded to observe the tumor growth tendency, the tumor growth delay and the mice survival time, 2. The incidence of lung metastasis two weeks after the radiation was recorded. 3. The incidence of lung metastasis when the tumor volume was four times as large as that at the beginning of radiation was recorded. Results: The adenovirus mediating Smad 7 gene expression regulated by irradiation via Egr-1 in C57BL mice implanted with Lewis lung cancer was able to inhibit the progression of the primary tumor and prolong the survival of the mice significantly as compared with the control group (P 0.05). Conclusions: The gene expression of AD. Egr-Smad 7 regulated by radiation is not risky in promoting the local progression and distant metastasis of Lewis lung cancer in mice. On the other hand, the gene expression of AD. Egr-Smad 7 regulated by radiation could inhibit the progression of the primary tumor and prolong the survival time of the mice significantly. It is safe, to some extent, of using AD

  3. Anti-tumor effect of adenovirus-mediated suicide gene therapy under control of tumor-specific and radio-inducible chimeric promoter in combination with γ-ray irradiation in vivo

    International Nuclear Information System (INIS)

    Objective: To detect the selective inhibitory effects of irradiation plus adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) suicide gene system using tumor-specific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse. Methods: Recombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radio-inducible CArG elements was constructed. A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA. The change of tumor volume and tumor growth inhibiting rate, the survival time of nude mice, as well as histopathology of xenograft tumor and normal tissues were evaluated. Results: Thirty one days after the treatment, the relative tumor volumes in the negative, adenovirus therapy, irradiation, and combination groups were 49.23±4.55, 27.71±7.74, 28.53±10.48 and 11.58±3.23, respectively.There was a significantly statistical difference among them (F=16.288, P<0.01).The inhibition effect in the combination group was strongest as compared with that in other groups, and its inhibition ratio was 76.5%. The survival period extended to 43 d in the combination group, which showed a significantly difference with that in the control group (χ2=18.307, P<0.01). The area of tumors necrosis in the combination group was larger than that in the other groups, and the normal tissues showed no treatment-related toxic effect in all groups. However, multiple hepatocellular carcinoma metastases were observed in the liver in the control group, there were a few metastases in the monotherapy groups and no metastasis in the combination group. Conclusions: Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong median

  4. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    Science.gov (United States)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  5. Adenovirus-mediated Transfer of p53 and p16 Inhibiting Proliferating Activity of Human Bladder Cancer Cell EJ in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    朱朝辉; 邢诗安; 林晨; 曾甫清; 鲁功成; 付明; 张雪艳; 梁萧; 吴旻

    2002-01-01

    Summary: To evaluate the effects of adenovirus (Ad)-mediated transfer of p53 and p16 on humanbladder cancer cells EJ, EJ were transfected with Ad-p53 and Ad-p16. Cell growth, morphologi-cal change, cell cycle, apoptosis were measured using MTT assay, flow gytometry, cloning forma-tion, immunocytochemical assays. Ad-p16 or Ad-p53 alone could inhibit the proliferating activityof EJ cells in vitro. Ad-p53 could induce apoptosis of partial EJ cells. G1 arrest was observed 72 hafter infection with Ad-p16, but apoptosis was not obvious. The transfer of Ad-p16 and Ad-p53could significantly inhibit the growth of EJ cells, decrease the cloning formation rate and induceapoptosis of large number of EJ cells. The occurrence time of subcutaneous tumor was delayed andthe tumor volume in 4 weeks was diminished by using Ad-p53 combined with Ad-p16 and the dif-ference was significant compared with using Ad-p53 or Ad-p16 alone. It was suggested that thetransfer of wild-type p53 and p16 could significantly inhibit the growth of human bladder cancer invitro and in vivo.

  6. Effects of recombinant adenovirus-mediated hypoxia-inducible factor-1alpha gene on proliferation and differentiation of endogenous neural stem cells in rats following intracerebral hemorrhage

    Institute of Scientific and Technical Information of China (English)

    Zhen Yu; Li-Fen Chen; Ling Tang; Chang-Lin Hu

    2013-01-01

    Objective:To investigate the effects of adenovirus(Ad)-mediated hypoxia-inducible factor-1alpha(HIF-1α) gene on proliferation and differentiation of endogenous neural stem cells(NSCs) in rats following intracerebral hemorrhage(ICH) and the underlying mechanisms.Methods:A total of120 specific pathogen-free, adult, maleSprague-Dawley rats were included in this study.After establishment ofICH models in rats,PBS,Ad, orAd-HIF-1αwas administered via the ischemic ventricle.On the1st,7th,14th,21st and28th d afterICH, rat neurological deficits were scored, doublecortin(DCX) expression in the subventricular zone cells was detected by immunohistochemical staining, and5-bromo-2'-deoxyuridine(BrdU)-,BrdU/DCX-, andBrdU/glial fibrillary acidic protein-positive cells in the subventricular zone were counted using immumofluorescence method amongPBS,Ad, andAd-HIF-1α groups.Results:On the7th, 14th,21st and28th d afterICH, neurological deficit scores in theAd-HIF-1α group were significantly lower than in thePBS andAd groups(P<0.05).In theAd-HIF-1α group,DCX expression was significantly increased on the7th d, peaked on the14th d, and then gradually decreased.In theAd-HIF-1α group,BrdU-positive cells were significantly increased over time course, and significant difference inBrdU-positive cell counts was observed when compared with thePBS andAd groups at each time point(P<0.01 or0.05).On the7th,14th,21st and28th d after ICH, the number ofDCX-,BrdU-,BrdU/DCX-, andBrdU/DCX-positive cells in theAd-HIF-1α group was significantly greater than in thePBS andAd groups(P<0.05).Conclusions:HIF-1α gene can promote the proliferation, migration and differentiation of endogenous neural stem cells afterICH, thereby contributing to neurofunctional recovery afterICH.

  7. THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

    Institute of Scientific and Technical Information of China (English)

    SONG; Wen-gang

    2001-01-01

    [1]Meyer Zum Buschenfelde C, Cramer S, Trumpfheller C, et al. Trypanosoma cruzi induces strong IL-12 and IL-18 gene expression in vivo: correlation with interferon-gamma (IFN-gamma) production [J]. Clin Exp Immunol 1997; 110:378.[2]Tominaga K, Yoshimoto T, Torigoe K, et al. IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells [J]. Int Immunol 2000; 12:151.[3]Takeda K, Tsutsui H, Yoshimoto T, et al. Defective NK cell activity and Th1 response in IL-18-deficient mice [J]. Immunity 1998; 8:383.[4]Tomura M, Zhou XY, Maruo S, et al. A critical role for IL-18 in the proliferation and activation of NK1.1+ CD3- cells [J]. J Immunol 1998; 160:4738.[5]Okamura H, Kashiwamura S, Tsutsui H, et al. Regulation of interferon-gamma production by IL-12 and IL-18 [J]. Curr Opin Immunol 1998; 10:259.[6]Osaki T, Hashimoto W, Gambotto A, et al. Potent antitumor effects mediated by local expression of the mature form of the interferon-gamma inducing factor, interleukin-18 (IL-18) [J]. Gene Ther 1999; 6:808.[7]Dinarello CA. IL-18: A TH1-inducing, proinflammatory cytokine and new member of the IL-1 family [J]. J Allergy Clin Immunol 1999; 103:11.[8]Matsui K, Yoshimoto T, Tsutsui H, et al. Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells [J]. J Immunol 1997; 159:97.[9]Akira S. The role of IL-18 in innate immunity [J]. Curr Opin Immunol 2000; 12:59.[10]Lauwerys BR, Garot N, Renauld JC, et al. Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18 [J]. J Immunol 2000; 165:1847.[11]Micallef MJ, Yoshida K, Kawai S, et al. In vivo antitumor effects of murine interferon-gamma-inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites [J]. Cancer Immunol Immunother 1997; 43:361.[12]Micallef MJ, Tanimoto T

  8. Growth Suppression of Human Lung Cancer Cells and Implanted Tumors by Adenovirus-mediated Transfer of the PTEN Gene

    Institute of Scientific and Technical Information of China (English)

    陈志雄; 杨炯

    2010-01-01

    This study examined the effects of a recombinant adenovirus Ad-PTEN-EGFP on the proliferation of A549 cells,a human lung carcinoma cell line,in vitro and on the growth of the implanted tumors in the nude mice in vivo,explored the underlying mechanisms and evaluated the in vitro transfection efficiency of Ad-PTEN-EGFP into A549 cells.The expression of Ad-PTEN-EGFP in the A549 cells was determined.The proliferation and the apoptosis rates of the A549 cells with Ad-PTEN-EGFP transfection or not was detected by...

  9. 制备源自HBsAg基因修饰树突状细胞的外切体%Generation of exosomes derived from adenovirus-mediated HBsAg gene-modified dendritic cells

    Institute of Scientific and Technical Information of China (English)

    杨静悦; 高琳; 付蓉; 薛妍; 刘文超

    2012-01-01

    Objective: To obtain exosomes derived from adenovirus - mediated HBsAg gene - modified dendritic cells. Methods: Full length HBsAg cDNAs were cloned into shuttle2 vector. The HBsAg gene fragments resulted from the - S digested with PI - See and I - Ceu were linked to the linear adeno - X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte - derived dendritic cells. The exosomes were isolated from superna-tant of transfected DCs. Transmission electron microscopy was used to observe their structures. The expressions of several proteins were investigated by flow cytometry. Results: The shuttle2 - S showed that band with 630 bp by di-gested with PI - See and I - Ceu, HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and pre-dictive fragments proved by restriction enzyme digestion analysis were exhibited. CPE appear 10 after days HEK293 cells transfected AdVHBsAg. Application of the isolation procedure to transfected DCs revealed exosome vesicles by transmission electron microscopy. Protein analysis by Western blot was performed and revealed that the costimulatory molecule CD86,CD83 and HBsAg was detectable. Conclusion; The exosomes derived from HBsAg - DC may be a tool of the HBV related hepatocellular carcinoma immunotherapy.%目的:制备一种新型负载HBsAg基因的外切体(exosome)瘤苗,并探讨其生物学特性、免疫学功能.方法:运用分子克隆和病毒载体转染HBsAg基因构建AdVHBsAg-DC肝癌瘤苗,采用流式细胞术鉴定转染基因表达;提取exosome;以透射电镜观察、Western blot法鉴定exosome.结果:构建的重组AdVHBsAg腺病毒载体,经PCR和酶切鉴定,结果显示HBsAg基因片段已正确插入腺病毒载体中.包装的腺病毒载体具有良好的感染性,可以在293细胞中形成病毒颗粒.提取的exosome在透射电镜下可观察到直径为50-100nm

  10. Adenovirus-mediated human endostatin gene delivery in the treatment of mouse melanoma%腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

    Institute of Scientific and Technical Information of China (English)

    曹瑞华; 廖万清; 温海; 刘翠杰

    2009-01-01

    目的 探讨腺病毒载体介导的内皮抑素基因(Ad-mES)在体外和体内的生物学活性.方法 不同感染复度(MOI)的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响.观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达.电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况.结果 腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%.靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强.瘤细胞接种后第8天,各组成瘤率100%.开始出现小鼠死亡的最早日:PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天.结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长(P<0.05),肿瘤体积增长减慢(P<0.05).%Objective To observe the bioactivity of adenovirus-mediated human endostatin gene in vivo and in vitro.Methods B16F10 melanoma cells and human endothelial cells(ECV 304)were both transfected with recombinant adenovirus containing green fluorescent protein(Ad-GFP)or human endostatin gene (Ad-mES) at various multiplicity of infection(MOI).Then,the expression of endostatin gene was detected by RT-PCR,and the growth of cells by MTT assay.B16F10 cells were inoculated into the back of mice to establish melanoma models,which were classified into treated groups intratumorally injected with Ad-mES once (single Ad-mES group) or repeatedly(repetitive Ad-mES group)with an interval of 7 days,and control groups intratumorally injected with Ad-GFP (Ad-GFP group)or phosphate buffred solution (PBS group).Subsequently,the growth of tumors was

  11. Adenovirus-mediated Expression of both Antisense Ornithine Decarboxylase and S-adenosylmethionine Decarboxylase Inhibits Lung Cancer Cell Growth

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Xianxi LIU; Bing ZHANG; Qifeng SUN; Dongfeng SUN

    2007-01-01

    Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector (named Ad-ODC-AdoMetDCas). To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC,the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector.Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G1 phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.

  12. Adenovirus-mediated neurotrophin-3 gene can over-express neurotrophin-3 in the motoneurons located at ventral horn of rat spinal cord%腺病毒介导的神经营养素-3基因能够在大鼠脊髓前角运动神经元内过表达神经营养素-3

    Institute of Scientific and Technical Information of China (English)

    陈元峰; 曾湘; 曾园山

    2011-01-01

    Objective To observe whether adenovirus-mediated neurotrophin-3 (NT-3) gene could over-express neu-rotrophin-3 in the motoneurons located at ventral hom of rat spinal cord, and derived efferent fibers of sciatic nerve. Methods NT-3 gene recombination adenovirus with green fluorescent protein (GFP) gene (report gene) were injected into the sciatic nerve. NT-3 overexpression of motoneurons located at ventral hom of spinal cord were observed under the fluorescent microscope, using immunofluorescence histochemistal staining technique, seven days after injecting the gene recombination adenovirus. Results GFP positive labeling cells were observed on cross sections of L, and L5 spinal cord segments in the animals of the GFP express (the control group) and NT-3+GFP express groups. In the NT-3+GFP express group, NT-3 positive labeling cells were observed also in L4 and L5 spinal cord segments. These cells were merged with GFP positive labeling cells, and were ventral horn's motoneurons over-expressing NT-3. Compared with the morphous of ventral horn's motoneurons in the GFP express group, motoneurons overexpress-ing NT-3 showed more branching processes in the NT-3+GFP express group. Conclusion Adenovirus-mediated NT-3 gene can over-express neurotrophin-3 in the motoneurons located at ventral hom of rat spinal cord and derived efferent fibers of sciatic nerve. The finding provides an initial experimental data for utilizing further a strategy of NT-3 gene therapy to repair experimental spinal cord injury.%目的 观察腺病毒介导的神经营养素-3 (NT-3)基因在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元的过表达.方法 在坐骨神经内直接注射含有绿色荧光蛋白(GFP)基因(报告基因)的NT-3基因重组腺病毒(Ad-NT-3-GFP),7d后应用免疫荧光组织化学染色技术,在荧光显微镜下观察脊髓前角运动神经元的NT-3过表达.结果 GFP表达组(对照组)和NT-3加GFP表达组两组动物的L4和L5脊髓段横

  13. A study on the ectopic cartilage formation of adipose-derived stem cells by adenovirus-mediated HIF-1α gene transfection%腺病毒介导HIF-1α修饰的脂肪源性干细胞异位成软骨的研究

    Institute of Scientific and Technical Information of China (English)

    潘玮敏

    2011-01-01

    目的 探讨重组腺病毒介导的低氧诱导因子-1α (HIF-1α)基因转染对脂肪源性干细胞(adipose-derived stem cells,ASCs)异位成软骨的作用.方法 构建能介导HIF-1α基因转染和表达的腺病毒载体(Ad-HIF-1α-GFP),转染体外培养的ASCs,RT-PCR检测其HIF-1α基因的表达,同时检测转染后细胞II型胶原的表达.随后将纤维蛋白胶分别与Ad-GFP-ASCs和Ad-HIF-1α-GFP-ASCs复合种植在裸鼠皮下,4周后行HE染色和甲苯胺蓝(Toluidine blue)染色进行观察.结果 荧光显微镜观察证实Ad-HIF-1α-GFP-ASCs的转染效率可达到80%;RT-PCR检测结果显示Ad-GFP-ASCs组微弱表达HIF-1α,Ad-HIF-1α-GFP-ASCs组HIF-1α基因则高表达,且Ad-HIF-1α-GFP-ASCs组II型胶原表达量明显高于Ad-GFP-ASCs组(P<0.05);形态学染色表明Ad-HIF-1α-GFP转染的ASCs主要为圆形,细胞伸展比率较低,但细胞数目较多,且细胞分泌较多的蛋白多糖.结论 腺病毒介导的HIF-1α可诱导ASCs向软骨细胞分化.%Objective To investigate the effect of ectopic cartilage formation of adipose-derived stem cells (ASCs) by adenovirus-mediated HIF-1α gene transfection.Methods Adenoviral vector was constructed with full length human HIF-1α gene and Ascs was transfected in vitro.The expressions of HIF-1α gene and collagen Ⅱ gene were detected by RT-PCR in transfected group.Then Fibrin glue and those composites mixed with Ad-GFP-ASCs and AdHIF-1α-GFP-ASCs respectively were transplanted into those naked mice.Those samples were acquired after 4 weeks and were tested by H&E and Toluidine blue staining.Results Confirmed by fluorescence microscope, the transfection efficiency of Ad-HIF-1α-GFP-ASCs was 80%.The expression of HIF-1α gene was positive in the Ad-HIF-1α-GFPASCs group and negative in the Ad-GFP-ASCs group according to the RT-PCR test results, and the relative value of the expression of collagen Ⅱ mRNA had significant difference between the two groups (P<0.05).Histology

  14. Adenovirus-mediated transfer of hepatocyte growth factor gene to human dental pulp stem cells under good manufacturing practice improves their potential for periodontal regeneration in swine

    OpenAIRE

    Cao, Yu; Liu, Zhenhai; Xie, Yilin; Hu, Jingchao; WANG Hua; Fan, Zhipeng; Zhang, Chunmei; Wang, Jingsong; Wu, Chu-Tse; Wang, Songlin

    2015-01-01

    Introduction Periodontitis is one of the most widespread infectious diseases in humans. We previously promoted significant periodontal tissue regeneration in swine models with the transplantation of autologous periodontal ligament stem cells (PDLSCs) and PDLSC sheet. We also promoted periodontal tissue regeneration in a rat model with a local injection of allogeneic bone marrow mesenchymal stem cells. The purpose of the present study is to investigate the roles of the hepatocyte growth factor...

  15. SYNERGISTIC EFFICACY OF ADENOVIRUS-MEDIATED bcl-Xs GENE THERAPY AND TOPOTECAN IN OVARIAN CANCER CELL%bcl-Xs基因转移与羟基喜树碱对卵巢癌细胞 生长抑制的协同效应

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To observe the presence of synergistic efficacy between adenovirus mediated bcl-Xs (Adv-bcl-Xs) gene therapy and chemotherapy on ovarian cancer cell. Methods NuTu-19 cells were infected by different titers of Adv-bcl-Xs and were treated with topotecan at the same time. Cell proliferation was measured 3 days later by MTT. Graphical representations of the statistical analyses recorded their interaction in tumor cells. Results The statistical results and graphical representations of the statistical modeling showed that the synergistic antiproliferative activity was present (P<0.01). Conclusion There were synergistic efficacies between Adv-bcl-Xs gene therapy and Topotecan on ovarian cancer cell.%目的 用复制缺陷型腺病毒介导bcl-Xs(Adv-bcl-Xs)对卵巢癌细胞作基因转移,联合使用羟基喜树碱,观察它们对卵巢癌细胞产生的生长抑制协同效应。方法 用不同浓度的Adv-bcl-Xs感染卵巢癌细胞株NuTu-19,同时联合使用不同浓度的羟基喜树碱。3天后,用噻唑蓝法检测各实验组之存活细胞。统计学软件分析结果并作图。结果 Adv-bcl-Xs与羟基喜树碱联合使用同它们单独作用相加效应比较,对卵巢癌细胞生长抑制效果明显增强(P<0.01)。结论 Adv-bcl-Xs与羟基喜树碱联合使用,对卵巢癌细胞生长抑制存在协同效应。

  16. 腺病毒介导荧光素酶报告基因感染间充质干细胞的研究%Infection with adenovirus-mediated luciferase reporter gene in mesenchymal stem cells and bioluminescence imaging

    Institute of Scientific and Technical Information of China (English)

    王一帆; 夏睿; 郭玉林; 郜发宝

    2013-01-01

    目的 构建携带萤火虫荧光素酶(Luc)报告基因的腺病毒载体(Ad-Luc),研究其感染大鼠骨髓间充质干细胞(BMSC)后的体内外生物发光成像.方法 从psiCHECK-2质粒中用PCR扩增Luc基因,克隆入腺病毒穿梭载体pShuttle-CMV后行Nhe Ⅰ/Xba Ⅰ双酶切和测序鉴定.重组腺病毒穿梭载体与骨架载体pAdeno同源重组并包装纯化后,测定其病毒滴度.用重组Ad-Luc感染BMSC,行体外生物发光成像确定最佳感染复数(MOI),并采用曲线拟合回归分析生物发光强度与MOI的关系.以锥虫蓝染色法评价细胞活力变化,计算细胞存活率.将转染后BMSC(1×106个)植入SD大鼠前肢肌肉内,行体内生物发光成像.细胞存活率组间比较采用两因素重复测量资料方差分析.结果 经酶切和测序鉴定证明,Ad-Luc构建成功,病毒滴度为1×1010空斑形成单位(PFU)/ml.体外生物发光检测结果显示最佳MOI值为50,Ad-Luc可高效感染BMSC,使其表达Luc,且拟合曲线示细胞生物发光强度随MOI增加而增强(R2 =0.98).转染组和未转染组细胞培养1、3、5、7d时,细胞存活率分别为(92.5±2.3)%与(94.1±1.8)%、(91.4±0.9)%与(92.7±2.0)%、(92.1±1.6)%与(93.3±2.4)%、(91.9±1.5)%与(93.0±3.1)%,2组间细胞活力的差异无统计学意义(F=4.38,P>0.05).体内生物发光成像结果示BMSC移植1、3、7d后仍有存活,但随时间延长,生物发光信号逐渐减弱.结论 Luc报告基因通过腺病毒载体成功转入BMSC,实现了光学报告基因成像对移植干细胞的示踪.%Objective To construct adenovirus vector containing firefly luciferase reporter gene (AdLuc) and infect bone marrow mesenchymal stem cells (BMSC),then to take bioluminescence imaging in vitro and in vivo for identification.Methods The luciferase gene was amplified with PCR from psiCHECK-2 plasmid and cloned into the adenoviral shuttle vector (pShuttle-CMV).It was confirmed by Nhe Ⅰ/Xba Ⅰ digestion and sequencing

  17. Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

    OpenAIRE

    Hiroshi Ishida; Radharaman Ray; Jack Amnuaysirikul; Keiko Ishida; Prabhati Ray

    2012-01-01

    Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of N...

  18. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Directory of Open Access Journals (Sweden)

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  19. Adenovirus mediated angiostatin gene therapy for ovarian cancer: experiment with nude mice%重组腺病毒载体介导血管抑素基因治疗裸鼠卵巢癌的实验研究

    Institute of Scientific and Technical Information of China (English)

    贾长茹; 杨树艳; 韩世愈; 孙蕾

    2008-01-01

    Objective To built an expression vector of angiostatin (AG) gene with recombinated replication defective adenovirus and investigate the therapeutic effect of human AG gene on ovarian cancer. Methods (1) Human AG K ( 1-3 ) cDNA was inserted into the vector pShuttle to build the recombinant plasmid pShttle-AG ( K1-3 ). pAdeno-X-AG (K1-3) was built by double-cut and recombinated pShttle-AG (K1-3) to vector pAdeno-X, and then recombinant adenovirus was finally prepared by transinfection of pAdeno-X-AG (K1-3) into to the human embryo kidney cells of the line 293. (2) Human ovarian cancer cells of the line SKOV3 were inoculated subcutaneously into nude mice of the line BALB/c nu/nu to establish model of human ovarian cancer. Then the mice were randomly divided into 3 groups to be injected with Ad = AG (K1-3), Ad-LacZ, or phosphate buffered saline (PBS) around the cancer every 5 days. The tumor size was measured every 5 days to calculate the tumor volume and tumor inhibition rate. Three days after the last injection the mice were killed. The tumor tissues, livers, and kidneys of the mice underwent imunohistochemistry to calculate the microvessel density (MVD) and expression of vessel endothelial growth factor (VEGF) and AG. Results The tumor volume and weight of the Ad-AG ( K1-3 ) group were significantly less than those of the PBS and Ad-LacZ groups ( all P 0. 05). The expression levels of CD34 and VEGF of the Ad-AG( K1-3 ) group were both significantly lower than those of the PBS and Ad-LacZ groups (all P 0. 05 ). Conclusion Human angiostatin mediated by adenovirus suppresses the angiogenesis and the growth of human ovarian cancer in the nude mice model, which suggests that it is promising in clinical application.%目的 构建携带血管抑素(AG)基因K(1-3)重组复制缺陷型腺病毒表达载体,研究腺病毒介导的人血管抑素基因对卵巢癌的治疗作用.方法 (1)将人血管抑素K(1-3)cDNA插入穿梭载体pShuttle产生重组质粒pShttle-AG(K1

  20. Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480

    Institute of Scientific and Technical Information of China (English)

    Ze-Qun sun; Chang-Sheng Deng; Shao-Yong Xu; Yong Du

    2008-01-01

    AIM: To explore the antitumor bioactivity of adenovirus-mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480.METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber end SW480 tumor cell growth inhibition in vitro and in vivo.RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ed-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited celt migration.CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.

  1. Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to γ-irradiation

    Institute of Scientific and Technical Information of China (English)

    Xin-li LI; Qing-hui MENG; Sai-jun FAN

    2009-01-01

    Aim:An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa ceUs using adenovirus-mediated UHRF1 gene transfer (Ad5-UHRF1). Methods: Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA (siRNA).Results: Increased expression of UHRF1 by AdS-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRFl-mediated radioresistance. Conclusion: These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.

  2. 腺病毒介导CDglyTK双自杀基因系统对裸鼠皮下移植瘢痕疙瘩的治疗作用%Effects of recombinant adenovirus-mediated double suicide genes on implanted human keloid: experiment with athymic mice

    Institute of Scientific and Technical Information of China (English)

    徐斌; 刘振中; 张敬; 宗宪磊; 蔡景龙

    2008-01-01

    目的 探讨由大肠杆菌胞嘧啶脱氨酶(CD)基因/5-氟胞嘧啶(5-Fc)和单纯疱疹病毒胸苷激酶(HSV-TK)基因/丙氧鸟苷(GCV)基因治疗系统整合形成的腺病毒介导CDgly/TK双自杀基因系统对瘢痕疙瘩的治疗作用及其机制.方法 采用皮下移植保留表皮的入瘢痕疙瘩组织块的方法建立瘢痕疙瘩裸鼠模型,术后第7天将20只模型裸鼠分4组,每组5只.A组瘢痕内注射生理盐水;B组瘢痕内注射生理盐水+腹腔注射5-Fc和GCV;C组瘢痕内注射自行构建的莆组CDglyTK双自杀基因腺病毒(CDgly/TK);D组瘢痕内注射CDgly/TK+腹腔注射5-Fc和GCV;用药持续18 d.术后2、7(用药前)、14、21、28、35、42 d测量各组瘢痕疙瘩组织块体积;术后42 d取出瘢痕疙瘩组织块,HE染色进行组织学检查,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法检测成纤维细胞凋亡情况,免疫组织化学染色检测Bcl-2、Bax蛋白质的表达.结果 用药前和用药后7、14、21、28、35 d,D组瘢痕疙瘩组织块体积(mm3)分别为173±5、172±5、147±5、125±6、112±7和84±9,从用药后14 d开始明显缩小(均P<0.05);而其他3组瘢痕疙瘩组织块体积均明显增大,从用约后7 d开始各时点测得的体积均明显大于D组(均P<0.05).D组瘢痕疙瘩组织中有大量小鼠组织细胞浸润,胶原结构破坏和成纤维细胞凋亡明显重于其他3组,Bcl-2蛋门质表达明显弱于而Bax蛋白质表达明显强于其他3组.结论 腺病毒介导的CDglyTK双自杀基因系统在瘢痕疙瘩裸鼠模型中对瘢痕疙瘩产生治疗作用,诱导成纤维细胞凋亡足其主要作用机制.%Objective To detect the effects of the recombinant adenovirus-mediated double suicide genes constructed by Escherichia coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) and herpes simplex virus-thymidine kinase (HSV-TK)/ganiclovir (GCV)-CDglyTK on implanted human keloids and mechanisms thereof.Methods Twenty nude mice were

  3. Adenovirus-mediated expression of Tob1 sensitizes breast cancer cells to ionizing radiation

    Institute of Scientific and Technical Information of China (English)

    Yang JIAO; Chun-min GE; Qing-hui MENG; Jian-ping CAO; Jian TONG; Sai-jun FAN

    2007-01-01

    Aim: To investigate the effect of the Tobl gene, a member of the Transducing Molecule of ErbB2/B-cell Translocation Ggene (TOB/BTG) family, by using the adenovirus-mediated expression of Tob 1 on radiosensitivity in a human breast cancer cell line MDA-MB-231. Methods: Cell survival was determined by clonogenic assay. Apoptosis was evaluated by DNA fragmentation gel electro-phoresis and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Protein expression was analyzed by Western blot assay and DNA repair was measured by a host cell reactivation assay. Results: We demonstrated that pre-irradiation treatment with Ad5-Tob 1 significantly increased radiosensitivity,accompanying the increased induction of apoptosis and the repression of DNA damage repair. Furthermore, Ad5-Tob 1-mediated radiosensitivity correlates with the upregulation of the pro-apoptotic protein Bax and the downregulation of several DNA double strand break repair proteins, including DNA-dependent protein kinases, Ku70 and Ku80, and X-ray-sensitive complementation group 4.Conclusion: Tobl, as a new radiosensitizer, is a new target in the radiotherapy of breast cancer via increasing apoptosis and suppressing DNA repair.

  4. Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Junliang Li; Xinke Xu; Shanyi Zhang; Meiguang Zheng; Zhonghua Wu; Yinlun Weng; Leping Ouyang; Jian Yu; Fangcheng Li

    2012-01-01

    In this study, we investigated the effects of adenovirus-mediated transfection of PC12 cells with glucose transporter 3 after ischemic injury. The results of flow cytometry and TUNEL showed that exogenous glucose transporter 3 significantly suppressed PC12 cell apoptosis induced by ischemic injury. The results of isotopic scintiscan and western blot assays showed that, the glucose uptake rate was significantly increased and nuclear factor kappaB expression was significantly decreased after adenovirus-mediated transfection of ischemic PC12 cells with glucose transporter 3. These results suggest that adenovirus-mediated transfection of cells with glucose transporter 3 elevates the energy metabolism of PC12 cells with ischemic injury, and inhibits cell apoptosis.

  5. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

    OpenAIRE

    Wang, Bing; Li, Juan; Xiao, Xiao

    2000-01-01

    Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too sm...

  6. Intravenous delivery of adenovirus-mediated soluble FLT-1 results in liver toxicity

    NARCIS (Netherlands)

    Mahasreshti, P.J.; Kataram, M.; Wang, Miao; Stockard, C.R.; Grizzle, W.E.; Carey, D.; Siegal, G.P.; Haisma, H.J.; Alvarez, R.D.; Curiel, D.T.

    2003-01-01

    Purpose: Vascular endothelial growth factor (VEGF) is a potent angiogenic agent and plays a major role in tumor growth and metastases. We have previously reported the locoregional (i.p.) delivery of adenovirus-mediated antiangiogenic soluble FLT-1 (sFLT-1; a naturally encoded potent VEGF antagonist)

  7. Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells%腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞*☆

    Institute of Scientific and Technical Information of China (English)

    尹承慧; 邱俊钦; 曾昭勋; 陈宗雄

    2013-01-01

      背景:骨髓间充质干细胞作为骨、软骨创伤缺损及退变修复的种子细胞越来越受到关注。目的:分析人骨形态发生蛋白2基因转染对白色封闭群大鼠(SD 大鼠)骨髓间充质干细胞的影响。方法:分离纯化 SD 大鼠骨髓间充质干细胞并体外扩增,通过腺病毒载体介导人骨形态发生蛋白2基因转染骨髓间充质干细胞,分别通过荧光显微镜观察荧光表达情况及蛋白质水平来测定转染后人骨形态发生蛋白2的表达,碱性磷酸酶定量测定鉴定成骨活性及 MTT 法评估人骨形态发生蛋白2转染对骨髓间充质干细胞的影响。结果与结论:从 SD 大鼠骨髓提取物中分离培养的细胞形态为梭形,呈铺路石状、漩涡状生长,经流式细胞仪检测及多项分化能力鉴定符合骨髓间充质干细胞的特征;经转染人骨形态发生蛋白2基因后,骨髓间充质干细胞表达人骨形态发生蛋白2、碱性磷酸酶;MTT 法检测转染人骨形态发生蛋白2基因后,骨髓间充质干细胞增殖能力明显增强(P <0.05)。说明人骨形态发生蛋白2基因转染骨髓间充质干细胞后可以持续、高效表达人骨形态发生蛋白2和碱性磷酸酶,在体外明显促进骨髓间充质干细胞的增殖。%BACKGROUND: Bone marrow mesenchymal stem cel s as the seed cel s for repair of bone and cartilage trauma and degeneration have been paid increasing attention. OBJECTIVE: To investigative the effects of human bone morphogenetic protein 2 gene transfection on Sprague-Dawley rat bone marrow mesenchymal stem cel s. METHODS: Sprague-Dawley rat bone marrow mesenchyal stem cel s were in vitro isolated, purified and amplified. Adenovirus-mediated human bone morphogenetic protein 2 was transfected into bone marrow mesenchymal stem cel s. CD90 and CD45 expression levels were tested by flow cytometry. The successful y packaged virus was transfected into bone marrow mesenchymal

  8. The effect of adenovirus-mediated recombinant Tum5 gene expression on Rhesus retinal vascular endothelial cells under high glucose%腺病毒介导Tum5重组基因对高糖刺激下恒河猴视网膜血管内皮细胞增生、迁移及管腔形成的影响

    Institute of Scientific and Technical Information of China (English)

    杨伟; 张琰; 孙靖; 韩倩; 贾育蓉; 张红

    2015-01-01

    Objective To observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation,migration and tubing of Rhesus RF/6A cell under high glucose.Methods To construct the adenovirus vector of recombinant Tum5 gene (rAd-TumS),and then infected RF/6A cell with it.The Flow Cytometry was used to detect the infection efficiency.RF/6A cells were divided into normal group,high glucose (HG)-control group (HG group),empty expression vector group (HG+rAd-GFP),and HG+rAd-Tum5 group.Western blot was used to detect the expression of TumS.The CCK-8 test was applied to detect the proliferation of RF/6A cell,the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose.The proliferation,migration and tubing of RF/6A were tested respectively by CCK-8 test,Transwell test and Matrigel test.Results The adenovirus vector of recombinant Tum5 gene was successfully constructed.The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry.The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell.The result of CCK-8 test,Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation,migration and tubing of the RF/6A cell (F=44.484,772.666,137.696;P<0.05).The comparison of each group indicated that the HG group was higher than normal group (P< 0.05).There were no statistical differences between HG group and HG+ rAd-GFP group (P>0.05).However,the HG+rAd-Tum5 group was less than HG group (P<0.05),and the same to HG+rAd-GFP (P<0.05).Conclusion The adenovirus vector of recombinant Tum5 gene can inhibit the proliferation,migration and tubing of RF/6A cell under high glucose.%目的 观察腺病毒介导Tum5重组基因对高糖刺激下恒河猴视网膜血管内皮细胞(RF/6A细胞)增生、迁移

  9. 重组腺病毒气管途径反复转染大鼠肺组织人类eNOS基因的转导效果%Efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase gene into lung tissue by repeated intratracheal transfection in rats

    Institute of Scientific and Technical Information of China (English)

    周锦; 曹惠鹃; 张铁铮; 金强; 王俊科

    2012-01-01

    Objective To investigate the efficiency of transduction of recombinant adenovirus-mediated human endothelial nitric oxide synthase (eNOS) into lung tissue by repeated intratracheal transfection in rats.Methods Sixty 3-4 month old male Wistar rats weighing 220-280 g were randomly divided into 2 groups:control group (group C,n =10) and eNOS gene transduction group (group T,n =50).The animals were anesthetized with intraperitoneal 10% chloral hydrate 35 mg/kg,tracheally intubated and mechanically ventilated (VT 2.5 ml,RR 60 bpm,FiO2 1.0).Recombinant adenovirus carrying human eNOS gene was given as gift by Professor Gerard from Texas University,Southwest Medical Center.In group T 50 μl of the recombinant adenovirus in concentration of 5 × 109 PFU/ml was instilled into trachea every 5 minutes for 12 times,while in group C equal volume of vector conservation solution was instilled instead.Pulmonary arterial blood samples were obtained at 2,5,7,14 and 21 d after intratracheal transfection (n =10 at each time point) for determination of serum NO concentration.The animals were immediately sacrificed after blood sample collection for determination of expression of eNOS protein in the lung tissue and RNA.The eNOS expression in the trachea,bronchus,lung,liver,spleen and kidney was detected by immuno-histochemistry.Results The serum NO concentrations were significantly higher at all time points in group T than in group C.The eNOS expression was detected in the epithelial cells of trachea and bronchi,and endothelial cells of alveoli and pulmonary blood vessels in group T but not in group C.eNOS expression was not detected in liver,spleen and kidney at 7 d after intratracheal transfection in group T.Conclusion Human eNOS gene mediated by recombinant adenovirus was transducted into rat lung tissue with normal enzyme activity by repeated intratracheal administration without being detected in distant organs.%目的 重组腺病毒气管途径反复转染大鼠肺组织人类内

  10. Beta-Adrenergic gene therapy for cardiovascular disease

    Directory of Open Access Journals (Sweden)

    Koch Walter J

    2000-10-01

    Full Text Available Abstract Gene therapy using in vivo recombinant adenovirus-mediated gene transfer is an effective technique that offers great potential to improve existing drug treatments for the complex cardiovascular diseases of heart failure and vascular smooth muscle intimal hyperplasia. Cardiac-specific adenovirus-mediated transfer of the carboxyl-terminus of the β-adrenergic receptor kinase (βARKct, acting as a Gβγ-β-adrenergic receptor kinase (βARK1 inhibitor, improves basal and agonist-induced cardiac performance in both normal and failing rabbit hearts. In addition, βARKct adenovirus infection of vascular smooth muscle is capable of significantly diminishing neointimal proliferation after angioplasty. Therefore, further investigation is warranted to determine whether inhibition of βARK1 activity and sequestration of Gβγ via an adenovirus that encodes the βARKct transgene might be a useful clinical tool for the treatment of cardiovascular pathologies.

  11. Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yuan; ZHAI Suo-qiang; SONG Wei; GUO Wei; ZHENG Gui-liang; HU Yin-yan

    2008-01-01

    Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possibl utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT-PCR analysis. The Hathl gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differenti-ate into hair cell-like cells when forced to express Hathl. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

  12. Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters.

    Science.gov (United States)

    Sallenave, J M; Xing, Z; Simpson, A J; Graham, F L; Gauldie, J

    1998-03-01

    This report describes the design and construction of three recombinant adenoviruses of serotype 5 (Ad5) expressing elafin (EL), also called elastase-specific inhibitor. Three promoters were chosen to drive the synthesis of elafin: the small (380 bp) human cytomegalovirus promoter (HCMV), the Ad2 major late promoter (MLP) and the mouse cytomegalovirus (MCMV) promoter. Human alveolar epithelial cells (A549), as well as rat and human primary pulmonary fibroblasts were infected with Ad5-HCMV-EL, Ad5-MLP-EL, Ad5-MCMV-EL and with the control Ad5-dl70/3. The MCMV promoter was the most efficient promoter in all cells studied. MLP was the least efficient promoter Intermediate between MCMV and MLP was HCMV which was able to induce significant amounts of elafin, particularly in human A549 cells. When compared in vivo in rat lungs, results were similar; MCMV was the only promoter which induced significant amounts of elafin as assessed by Northern blot analysis and ELISA, even with a low dose of virus (3 x 10(8) p.f.u.). Our data indicate that the MCMV promoter is the promoter of choice for the strong induction of adenovirus-mediated transgenes in the lung and suggest its suitability both in rodent experimental models and in humans for investigative and therapeutic purposes. PMID:9614555

  13. Panspermia and horizontal gene transfer

    Science.gov (United States)

    Klyce, Brig

    2009-08-01

    Evidence that extremophiles are hardy and ubiquitous is helping to make panspermia a respectable theory. But even if life on Earth originally came from space, biologists assume that the subsequent evolution of life is still governed by the darwinian paradigm. In this review we show how panspermia could amend darwinism and point to a cosmic source for, not only extremophiles but, all of life. This version of panspermia can be called "strong panspermia." To support this theory we will discuss recent evidence pertaining to horizontal gene transfer, viruses, genes apparently older than the Earthly evolution of the features they encode, and primate-specific genes without identifiable precursors.

  14. Construction of recombinant adenovirus vector containing AFP and generation of adenovirus-mediated AFP gene modified dendritic cells vaccine%含人AFP基因重组腺病毒载体的构建及其转染树突状细胞瘤苗的制备

    Institute of Scientific and Technical Information of China (English)

    杨静悦; 曹大勇; 刘文超; 斯小明

    2009-01-01

    Objective:To construct recombinant adenovirus vectors containing human AFP genes,and infect dendritic cell. Methods: Full length AFP cDNAs were subcloned into pIND vector,followed by being cloned into shuttle2 vector.The AFP gene fragments resulted from the shuttle2-AFP digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA.After packaged with HEK293 cells,the adenovirus expression vector was obtained.The plasmid pAdeno-AFP was identified by endonuclease and PCR.After dendritic cells were infected pAdeno-AFP,the surface molecules of pAdeno-AFP/DC were analysed by flow cytometry.AFP levels in culture supernatant of pAdeno-AFP/DC were measured by ELISA. Results: AFP gene in the inserted DNA of adeno-AFP was confirmed by PCR,and predictive fragments proved by restriction enzyme digestion analysis were exhibited.All the above results indicated that human AFP gene had been connected with pAdeno-X vectors correctly.The recombinant adenovirus vector of human AFP gene packaged in HEK293 cells,it will be used to introduce the target gene into dendritic cell.pAdeno-AFP/DC were able to upregulate CD1a,CD11c,CD80,CD86 and HLA-DR.And pAdeno-AFP/DC could secrete high level of AFP in vitro. Conclusion: The recombinant adenovirus vector of human AFP gene have been constructed successfully.The established AFP -DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy,and it will be the foundation of future clinical use of DC vaccine.%目的:构建含人AFP基因的腺病毒载体,体外转染树突状细胞,制备树突状细胞肝癌瘤苗.方法: 将AFP基因亚克隆到pIND 载体和Shuttle2载体中,构建穿梭载体Shuttle2-AFP.用PI-Sce Ⅰ和I-CeuⅠ双酶切后将所获AFP基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-AFP重组腺病毒载体.其后,用重组腺病毒载体转染HEK293细胞,包装腺病毒表达载体.通过酶切、PCR对腺病毒载体进行鉴定.包装好的重组病毒载体pAdeno-AFP体外

  15. Construction of Recombinant Adenovirus Carrying gfp Gene and Adenovirus-mediated GFP Expression in Human Vascular Smooth Muscle Cells%携带绿色荧光蛋白基因的重组腺病毒的构建及其在人血管平滑肌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    张蕾; 王家宁; 郭凌郧; 孔霞; 杨建业; 唐俊明; 黄永章; 郑飞

    2009-01-01

    目的:构建携带绿色荧光蛋白(Green Fluorescent Protein,GFP)基因的重组腺病毒质粒pAd-GFP,制备重组腺病毒Ad-GFP,并使GFP在血管平滑肌细胞中得到高效表达.方法:将线性化的穿梭质粒pRNAT-H1.1与腺病毒骨架质粒pAdeagy-1在感受态BJ5183内进行同源重组,并筛选出阳性重组子pAd-GFP;用Pac I酶切线性化pAdGFP,线性化的腺病毒质粒经脂质体转染AD293细胞,进行重组腺病毒的包装和扩增.采用CsCl密度梯度离心进行病毒浓缩和纯化.获得的腺病毒感染人血管平滑肌细胞(human vascular smooth muscle cell,hVSMC),观察其感染效率和GFP表达水平.结果:通过Pac I酶切证实腺病毒载体构建成功,包装出携带GFP基因的腺病毒,滴度达到4.5 × 1012pfu/mL,获得的腺病毒对hVSMC的感染效率约为100%.结论:利用细菌内同源重组方法成功地构建了携带绿色荧光蛋白的重组腺病毒,并能够在hVSMC细胞中高效地表达,为利用GFP作为报告基因的研究奠定了实验基础.%Obieaive To construct recombinant edvenovirus phsmid containing GFP gene and prepare recombinant adenovirus pAd-GFP and GFP will be efficiently expressed in human vascular smooth muscle cells(hVSMC).Methods pRNATH1.1/Adeno was linearized with Pme I,and transformed into ultracompletent BJ5183 containing pAdeasy-1,then reeombinant advenovirus pAd-GFP was constructed by homologous recombination in bacteria BJ5 183.The recombinant adenoviral plasmid DAd-GFP Was identified bv Pac I dizestion.Linearized DAd-GFP was transfeeted into AD293 cells with llposome to generate recombinant adenovirus particles which were purified and concentrated by Cesium cMoride(CsCl)density gradient eentrifugation.Adenovirus particles were used to infect hVSMC,and the infection efficiency and GFP expression were observed under inveaed phase-contrast microscope.Results The adenovirus vector WaS constructed successfully and adenoviruff encoding GFP gene Wag prepared with titers

  16. Horizontal gene transfer in chromalveolates

    Directory of Open Access Journals (Sweden)

    Bhattacharya Debashish

    2007-09-01

    Full Text Available Abstract Background Horizontal gene transfer (HGT, the non-genealogical transfer of genetic material between different organisms, is considered a potentially important mechanism of genome evolution in eukaryotes. Using phylogenomic analyses of expressed sequence tag (EST data generated from a clonal cell line of a free living dinoflagellate alga Karenia brevis, we investigated the impact of HGT on genome evolution in unicellular chromalveolate protists. Results We identified 16 proteins that have originated in chromalveolates through ancient HGTs before the divergence of the genera Karenia and Karlodinium and one protein that was derived through a more recent HGT. Detailed analysis of the phylogeny and distribution of identified proteins demonstrates that eight have resulted from independent HGTs in several eukaryotic lineages. Conclusion Recurring intra- and interdomain gene exchange provides an important source of genetic novelty not only in parasitic taxa as previously demonstrated but as we show here, also in free-living protists. Investigating the tempo and mode of evolution of horizontally transferred genes in protists will therefore advance our understanding of mechanisms of adaptation in eukaryotes.

  17. Transferring alien genes to wheat

    International Nuclear Information System (INIS)

    In broad terms an alien gene can be considered to be any gene transferred to wheat from a related species. As described above by Maan (section 7D) the genus Triticum contains a broad range of species, some of which cross readily with the cultivated tetraploid (T. Turgidum L.) or hexaploid (T. aestivum L.) wheats, and others only with great difficulty. In addition, wheat will also cross with species in a number of other genera including Agropyron, Elymus, Elytrigia (=Agropyron), Haynaldia, Hordeum, and Secale (Riley and Kimber, 1966; Knobloch, 1968; Feldman and Sears, 1981). In discussing the Triticum and Aegilops spp., the classification by Kimber and Sears, section SA-I, above, will be followed. For the Agropyron and related species the classification described by Dewey (1983) will be used. To avoid confusion, in referring to the literature the designations used by the authors will be given, followed by the new designation. The wild relatives of wheat are adapted to a broad range of environments and carry a large reservoir of useful genes (Zohary et al., 1969; Kerber and Dyck, 1973; Brezhnev, 1977; Feldman and Sears, 1981; Limin and Fowler, 1981; Sharma et aI., 1981; McGuire and Dvorak, 1981). Initially they were considered to be primarily sources of disease resistance, but more recently they have been recognized as potential sources of genes for high protein, cold tolerance, salt tolerance, drought tolerance, lodging resistance, early maturity, and even yield. Extensive screening of the wild relatives of wheat needs to be done before their useful genes can be fully utilized

  18. Horizontal gene transfer in the phytosphere

    NARCIS (Netherlands)

    Elsas, van J.D.; Turner, S.; Bailey, M.J.

    2003-01-01

    Here, the ecological aspects of gene transfer processes between bacteria in the phytosphere are examined in the context of emerging evidence for the dominant role that horizontal gene transfer (HGT) has played in the evolutionary shaping of bacterial communities. Moreover, the impact of the putative

  19. Translating Gene Transfer: A Stalled Effort

    OpenAIRE

    Greenberg, Alexandra J.; McCormick, Jennifer; Tapia, Carmen J.; Windebank, Anthony J.

    2011-01-01

    The journey of gene transfer from laboratory to clinic has been slow and fraught with many challenges and barriers. Despite the development of the initial technology in the early 1970s, a standard clinical treatment involving “gene therapy” remains to be seen. Furthermore, much was written about the technology in the early 1990s, but since then, not much has been written about the journey of gene transfer. The translational path of gene transfer thus far, both pitfalls and successes, can serv...

  20. Targeting Radiotherapy to Cancer by Gene Transfer

    OpenAIRE

    R. J. Mairs; Boyd, M.

    2003-01-01

    Targeted radionuclide therapy is an alternative method of radiation treatment which uses a tumor-seeking agent carrying a radioactive atom to deposits of tumor, wherever in the body they may be located. Recent experimental data signifies promise for the amalgamation of gene transfer with radionuclide targeting. This review encompasses aspects of the integration of gene manipulation and targeted radiotherapy, highlighting the possibilities of gene transfer to assist the targeting of cancer ...

  1. Adenovirus-mediated siRNA targeting CXCR2 attenuates titanium particle-induced osteolysis by suppressing osteoclast formation.

    Science.gov (United States)

    Wang, Chen; Liu, Yang; Wang, Yang; Li, Hao; Zhang, Ran-Xi; He, Mi-Si; Chen, Liang; Wu, Ning-Ning; Liao, Yong; Deng, Zhong-Liang

    2016-01-01

    BACKGROUND Wear particle-induced peri-implant loosening is the most common complication affecting long-term outcomes in patients who undergo total joint arthroplasty. Wear particles and by-products from joint replacements may cause chronic local inflammation and foreign body reactions, which can in turn lead to osteolysis. Thus, inhibiting the formation and activity of osteoclasts may improve the functionality and long-term success of total joint arthroplasty. The aim of this study was to interfere with CXC chemokine receptor type 2 (CXCR2) to explore its role in wear particle-induced osteolysis. MATERIAL AND METHODS Morphological and biochemical assays were used to assess osteoclastogenesis in vivo and in vitro. CXCR2 was upregulated in osteoclast formation. RESULTS Local injection with adenovirus-mediated siRNA targeting CXCR2 inhibited titanium-induced osteolysis in a mouse calvarial model in vivo. Furthermore, siCXCR2 suppressed osteoclast formation both directly by acting on osteoclasts themselves and indirectly by altering RANKL and OPG expression in osteoblasts in vitro. CONCLUSIONS CXCR2 plays a critical role in particle-induced osteolysis, and siCXCR2 may be a novel treatment for aseptic loosening. PMID:26939934

  2. ADENOVIRUS-MEDIATED WILD-TYPE P53 EXPRESSION SUPPRESSES GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji

    1998-01-01

    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  3. Combined adenovirus-mediated artificial microRNAs targeting mfgl2, mFas, and mTNFR1 protect against fulminant hepatic failure in mice.

    Directory of Open Access Journals (Sweden)

    Dong Xi

    Full Text Available Hepatitis B virus (HBV-related acute-on-chronic liver failure (ACLF has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2, FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA targeting mouse fgl2 (mfgl2 or both mFas and mTNFR1 on murine hepatitis virus (MHV-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA,which expresses miRNA against both mFas and mTNFR1 simultaneously,was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4% mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2% mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7% mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.

  4. 腺病毒介导多基因对大鼠脾淋巴细胞毒作用的影响%Effect of adenovirus-mediated multigenes on cytotoxicity of rat spleen lymphocyte in vitro

    Institute of Scientific and Technical Information of China (English)

    王征旭; 何振平; 吴祖泽

    2001-01-01

    Objective To investigate the changes of the cytotoxicity of ratspleen lymphocyte and the level of IL-2 secreted by human T lymphocyte after the induction of adenovirus-mediated multigenes (Ad-multigenes, containing p53, GM-CSF, B7-1, IL-2 genes). Methods After human lymphocytes of peripheral blood and tumor cells were cultured together, the level of IL-2 secreted by T lymphocytes was determined after they were stimulated by liver cancer cells with pre-transfer of Ad-multigenes in vitro by ELISA. The change of the immunogenicity of rat carcinosarcoma cell Walker 256 transduced with multigenes was studied by cytotoxicity assay of rat spleen lymphocytes. Results The level of IL-2 secreted by peripheral blood T lymphocytes was increased in vitro after the T cells were co-cultivated with Ad-multigene-transducted liver cancer cells. Stimulated by Ad-multigene-transducted Walker 256 cells, the cytotoxicity activities of rat spleen lymphocyte were significantly elevated. Conclusion The immunogenicity of rat carcinosarcoma cell Walker 256 is enhanced, and the IL-2 production level which was secreted by T lymphocyte is increased after the mediation of Ad-multigenes.%目的 研究含多基因(p53、GM-CSF、B7-1、IL-2)的重组腺病毒载体Ad-multigenes,对大鼠脾脏淋巴细胞毒作用的影响及对淋巴细胞分泌IL-2的刺激作用。方法 应用人外周血淋巴细胞和肿瘤细胞混合培养,分析导入目的基因的肝癌细胞系体外刺激人T淋巴细胞分泌IL-2的作用;利用大鼠脾淋巴细胞杀伤活性试验,分析导入目的基因的大鼠癌肉瘤Walker256细胞,其免疫原性的变化。结果 导入Ad-multigenes的肝癌细胞系体外刺激人外周血T淋巴细胞分泌IL-2的水平增加;导入Ad-multigenes的大鼠Walker256细胞,能增强大鼠脾脏淋巴细胞的杀亲本瘤细胞活性。结论 腺病毒介导多基因Ad-multigenes,能增强大鼠癌肉瘤Walker256细胞的免疫原性,和T细胞分泌IL-2的水平增加。

  5. Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Yi-zhen; ZHANG Jun; LIU Zeng-li; DU Shou-ying; SHEN Yong-mei

    2010-01-01

    Background The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaCIO4).The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P <0.001).Conclusion

  6. Adenovirus-mediated ING4 expression reduces multidrug resistance of human gastric carcinoma cells in vitro and in vivo.

    Science.gov (United States)

    Mao, Zong-Lei; He, Song-Bing; Sheng, Wei-Hua; Dong, Xiao-Qiang; Yang, Ji-Cheng

    2013-11-01

    Chemotherapy is the primary treatment for both resectable and advanced gastric carcinoma, yet multiple drug resistance (MDR) of gastric carcinoma remains a significant therapeutic obstacle. The development of novel strategies to reduce MDR in gastric carcinoma would yield a better outcome following chemotherapy. ING4, a member of the inhibitor of growth (ING) tumor-suppressor family, possesses antitumor and radiosensitization or chemosensitization effects in a variety of human cancers. The present study investigated the effects and possible mechanisms of action of adenovirus-mediated ING4 (AdVING4) on the reversion of human gastric carcinoma cell MDR in vitro and in vivo in nude mouse xenografts. The data showed that the expression of ING4 mRNA and protein was dramatically downregulated (or lost) in gastric carcinoma SGC7901/CDDP cells after CDDP-induced MDR phenotype and in the parental SGC7901 cells. AdVING4‑induced ING4 expression reversed MDR and induced apoptosis of SGC7901/CDDP cells in vitro and in vivo in the SGC7901/CDDP xenograft tumors. Furthermore, AdVING4 substantially downregulated the expression of MDR-related proteins P-gp and MRP1 and apoptosis‑related proteins Bcl-2 and survivin, but upregulated the expression of apoptosis-related protein Bax in the SGC7901/CDDP xenograft tissues. The reversion effects elicited by AdVING4 on gastric cancer cell MDR were closely associated with the downregulation of ATP-binding cassette transporters and activation of apoptotic pathways. Thus, these findings suggest that AdVING4 may be a feasible modulator for the MDR phenotype of gastric carcinoma cells. PMID:23969950

  7. Gene transfer to promote cardiac regeneration.

    Science.gov (United States)

    Collesi, Chiara; Giacca, Mauro

    2016-12-01

    There is an impelling need to develop new therapeutic strategies for patients with myocardial infarction and heart failure. Leading from the large quantity of new information gathered over the last few years on the mechanisms controlling cardiomyocyte proliferation during embryonic and fetal life, it is now possible to devise innovative therapies based on cardiac gene transfer. Different protein-coding genes controlling cell cycle progression or cardiomyocyte specification and differentiation, along with microRNA mimics and inhibitors regulating pre-natal and early post-natal cell proliferation, are amenable to transformation in potential therapeutics for cardiac regeneration. These gene therapy approaches are conceptually revolutionary, since they are aimed at stimulating the intrinsic potential of differentiated cardiac cells to proliferate, rather than relying on the implantation of exogenously expanded cells to achieve tissue regeneration. For efficient and prolonged cardiac gene transfer, vectors based on the Adeno-Associated Virus stand as safe, efficient and reliable tools for cardiac gene therapy applications.

  8. Adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

    Institute of Scientific and Technical Information of China (English)

    Dongxu Feng; Haopeng Li; Siyue Xu; Yu Liu; Xiaofei Hou

    2011-01-01

    Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA (shRNA) for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments (shRNA) were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector (carrying shRNA) and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells (PC12). Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49±0.68%, 95.08±1.79% and 96.60±1.14% at the mRNA level, and 72.89±2.17%, 58.83±1.15% and 59.88±0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

  9. Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hu Wang; Sheng-Bao Li; Qiang Tong; Guo-Jian Xie; Qing-Ming Wu

    2005-01-01

    AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2antisense RNA, and to explore its effects on liver cancer cell proliferation.METHODS: We studied the expression of COX-2 in 34cases of hepatocellular carcinoma (HCC) and SMMC7402and SMMC7721 by immunohistochemical technique.Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry.Cell proliferation was determined by colony-forming efficiency.RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06× 1012 PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line.The difference of apoptotic index between the Ad-AShcox2 group and control group was statistically significant(tcontrol group= 32.62 and tAd-LacZ= 10.93, P<0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and(33.6±4.24)%, respectively.CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

  10. Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

    Directory of Open Access Journals (Sweden)

    Zhao Yarui

    2011-01-01

    Full Text Available Abstract Background Sphingomyelin synthase 2 (SMS2 contributes to de novo sphingomyelin (SM biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. Methods The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR and protein level examination (SMS activity assay. Result We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2 or GFP cDNA (AdV-GFP. On day six after intravenous infusion of 2 × 1011 particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p Conclusions Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.

  11. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Ayesha; Ellenson, Lora Hedrick, E-mail: lora.ellenson@med.cornell.edu

    2011-07-01

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten{sup +/-} mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten{sup +/-} mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ER{alpha} as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  12. Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

    International Nuclear Information System (INIS)

    Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten+/- mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten+/- mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

  13. Viral Vectors for in Vivo Gene Transfer

    Science.gov (United States)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  14. Adenovirus-mediated expression of SSAT inhibits colorectal cancer cell growth in vitro

    Institute of Scientific and Technical Information of China (English)

    Hui SUN; Bin LIU; Ya-pei YANG; Chun-xiao XU; Yun-fei YAN; Wei WANG; Xian-xi LIU

    2008-01-01

    Aim: To construct a recombinant adenovirus that can express human spermidine/ spermine N1-acetyltransferase (SSAT) and detect its inhibitory effect on colorectal cancer cell growth in vitro. Methods: A 516 bp eDNA of SSAT was amplified and cloned into a pGL3-hTERT plasmid. The pGL3-hTERT-SSAT recombinant was digested, and the small fragment was cloned into the shuttle vector pAdTrack. The pAdTrack-hTERT-SSAT plasmids were recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the HEK293 packaging cells (transformed human embryonic kidney cells) after they were lin-earized by PacI. The process of adenovirus packaging and amplification was monitored by green fluorescent protein (GFP) expression. The SSAT protein levels were determined by Western blotting, and the intracellular polyamine con-tent was detected by reverse-phase high performance liquid chromatography. The MTS (3-(4, 5-dimethylthiaol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(-4-sulfophenyl)-2H-tetrazolium, inner salt) and colony-forming assays were used to analyze the gene transduction efficiency and effect on the growth of HT-29 and LoVo cells. A viable cell count was used to determine the cell growth with or without exogenous polyamines. Results: The GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting results demonstrated that Ad-hTERT-SSAT could increase the expres-sion of SSAT, and consequently, spermidine and spermine were reduced to low levels. The MTS and colony-forming assay results showed that HT-29 and LoVo cell growth were significantly inhibited, and the inhibitory effect could be partially reversed by exogenous spermidine and spermine. Conclusion: The successfully constructed recombinant adenovirus Ad-hTERT-SSAT could accelerate polyamine catabolism and inhibit the colorectal cell growth in vitro. It also has therapeutic potential in the treatment of colorectal cancer.

  15. Adenovirus-mediated delivery of p27KIP1 to prevent wound healing after experimental glaucoma filtration surgery

    Institute of Scientific and Technical Information of China (English)

    Jian-gang YANG; Nai-xue SUN; Li-jun CUI; Xiao-hua WANG; Zhao-hui FENG

    2009-01-01

    Aim: The aim of the study was to evaluate the outcome of adenovirus-mediated p27KIP1 (Ad-p27) expression on wound healing after filtration surgery and to investigate the inhibition of cell proliferation induced by Ad-p27. Methods: We constructed the adenovirus recombinant vector Ad-p27 and administered it to a rabbit model of glaucoma filtration surgery by subconjunctival injection; phosphate-buffered saline (PBS) and mitomycin C (MMC) were used as controis. Intraocular pressure (IOP), bleb scores, and anterior chamber depths were observed during a 28-d period. Histological examinations, fluorescence observations and Western blot analyses were evaluated.Results: Ad-p27 enhanced the surgical outcome and inhibited cell proliferation when compared with PBS. Bleb scores in the Ad-p27-treated eyes were higher than those in the PBS-treated eyes on d 7 (P<0.01), 14 (P<0.01) and 21 (P<0.05). Ond 28, IOP remained significantly decreased in the Ad-p27 group compared with the PBS group (P<0.05). However, no differences in bleb scores or IOPs were observed between the Ad-p27 and MMC groups. Histological analysis showed that total cell numbers were markedly reduced, and less scar tissue was observed at the surgical site in eyes treated with Ad-p27.The number of fibroblasts was decreased in Tenon's capsule in Ad-p27-treated eyes; however, a marked and diffuse signal from the green fluorescent protein (GFP) was observed in fibroblasts. Western blot analysis revealed a high level of p27KIP1 expression in conjunctival epithelium (P<0.01), relatively high expression in superficial scleral stroma (P<0.01), and low expression in corneal epithelium in the Ad-p27 group. Conclusions: Ad-p27 administration significantly improves the outcome of filtration surgery and inhibits postoperative proliferation in rabbit eyes. These findings suggest that p27KIP1 is a potential adjunctive agent for inhibition of wound heal-ing after filtration surgery.

  16. Rates of Lateral Gene Transfer in Prokaryotes

    NARCIS (Netherlands)

    Vos, Michiel; Hesselman, M.C.; Beek, te T.A.; Passel, van M.W.J.; Eyre-Walker, Adam

    2015-01-01

    Lateral gene transfer is of fundamental importance to the evolution of prokaryote genomes and has important practical consequences, as evidenced by the rapid dissemination of antibiotic resistance and virulence determinants. Relatively little effort has so far been devoted to explicitly quantifyi

  17. 体外骨髓基质干细胞中腺病毒介导的胶质细胞源性神经营养因子基因的表达及其生物学活性%Expression of adenovirus-mediated glial cell line-derived neurotrophic factor gene in bone marrow stromal cells in vitro and its biological activity

    Institute of Scientific and Technical Information of China (English)

    彭松林; 方煌; 赵红卫; 蔡卫东; 黎逢峰; 胡宁; 陈安民

    2005-01-01

    背景:骨髓基质干细胞(bone marrow stromal cells,BMSCs)是外源性目的基因的良好靶细胞,在脊髓损伤的修复中具有良好的应用前景.目的:观察重组腺病毒介导的胶质细胞源性神经营养因子(glial cellline-derived neurotrophic factor,GDNF)基因在体外培养的骨髓基质干细胞中的表达,并探讨其生物学活性.设计:以细胞为研究对象,对照观察性研究.单位:一所大学医院骨科实验室.材料:实验于2004-03/06在华中科技大学同济医学院附属同济医院骨科实验室完成.SD大鼠24只,雌雄不限,体质量(180±20)g.干预:用重组腺病毒载体Adv-GDNF感染体外培养的BMSCs,并与脊髓背根神经节共培养.免疫荧光化学的方法检测BMSCs中的GDNF的表达,提取细胞总RNA进行RT-PCR扩增GDNF基因,应用ELISA方法检测其培养上清中的GDNF含量,并通过与脊髓背根神经节共培养观测GDNF的活性.主要观察指标:主要结局:①RT-PCR.②免疫荧光结果.③GDNF的体外活性.次要结局:①BMSCs的培养与鉴定.②ELISA检测蛋白表达与时间的关系.结果:免疫荧光显示Adv-GDNF感染BMSCs 48 h后即有GDNF的表达,体外培养的BMSCs经Adv-GDNF转染后有GDNF的转录,其培养上清应用ELISA方法分析,在感染24 h后即有GDNF的表达,并可持续5~7 d的高峰.Adv-GDNF感染的BMSCs的培养液上清可以促进脊髓背根神经节大量轴突的生长.结论:Adv-GDNF基因可以在BMSCs中稳定、高效表达,其表达的GDNF具有促进轴突生长的活性,为GDNF基因治疗脊髓损伤的研究奠定了基础.%BACKGROUND: Bone marrow stromal cells(BMSCs) are the ideal gene target cells and will have a bright future in the gene therapy of spinal cord injury.OBJECTIVE :To detect the expression of glial cell line - derived neurotrophic factor(GDNF) gene after BMSCs were infected by adenovirus-medialed GDNF (Adv-GDNF) in vitro and to explore its biological activity.DESIGN: A randomized controlled trial

  18. Horizontal gene transfer and bacterial diversity

    Indian Academy of Sciences (India)

    Chitra Dutta; Archana Pan

    2002-02-01

    Bacterial genomes are extremely dynamic and mosaic in nature. A substantial amount of genetic information is inserted into or deleted from such genomes through the process of horizontal transfer. Through the introduction of novel physiological traits from distantly related organisms, horizontal gene transfer often causes drastic changes in the ecological and pathogenic character of bacterial species and thereby promotes microbial diversification and speciation. This review discusses how the recent influx of complete chromosomal sequences of various microorganisms has allowed for a quantitative assessment of the scope, rate and impact of horizontally transmitted information on microbial evolution.

  19. The use of alien gene transfers

    International Nuclear Information System (INIS)

    The present status of the gene transfers from alien species belonging to the sub-tribe Triticanae into wheat is reviewed, and the advantages and disadvantages of the different methods available for such transfers are examined. In general, the alien genes provide a high degree of resistance against a notably wide range of physiological races of wheat rusts, powdery mildew and other diseases. The alien resistance, like other sources of resistance, is known to break down for certain new races. This may happen more often when alien genes of resistance are widely incorporated in commercial cultivars and grown over large areas. So far, few of the available induced translocation stocks have contributed to the development of agronomically superior commercial cultivars, mainly due to the associated undesirable effects of the translocations on agronomic characters of the recipient variety. The deleterious effects appear in some genetic backgrounds and not in others. Extensive hybridization of translocation stocks with different genotypes has been emphasized by most investigators. Such programmes have led to the release of three commercial cultivars - 2 in Australia and 1 in the USA. On the other hand, spontaneous wheat-rye translocations carrying gene(s) for disease resistance have been unconsciously incorporated into several wheat cultivars, some of them are widely cultivated and were top in ranking based on grain yield. (author)

  20. Gene Transfer with Poly-Melittin Peptides

    OpenAIRE

    Chen, Chang-Po; Kim, Ji-Seon; Steenblock, Erin; Liu, Dijie; Rice, Kevin G.

    2006-01-01

    The 26 amino acid hemolytic melittin peptide was converted into a gene transfer peptide that binds to DNA and polymerized through disulfide bond formation. Melittin analogues were synthesized by addition of one to four Lys repeats at either the C or N-subterminal end along with terminal Cys residues. Melittin analogues were able to bind and polymerize on plasmids resulting in the formation of DNA condensates. In the absence of DNA, melittin analogues retained their red blood cell hemolytic po...

  1. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    Directory of Open Access Journals (Sweden)

    R. Thane Papke

    2015-05-01

    Full Text Available The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria.

  2. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    Science.gov (United States)

    Papke, R. Thane; Corral, Paulina; Ram-Mohan, Nikhil; de la Haba, Rafael R.; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  3. Horizontal gene transfer, dispersal and haloarchaeal speciation.

    Science.gov (United States)

    Papke, R Thane; Corral, Paulina; Ram-Mohan, Nikhil; Haba, Rafael R de la; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  4. Ultrasound and Microbubbles: Their Functions in Gene Transfer In Vitro

    Institute of Scientific and Technical Information of China (English)

    CHEN Yunchao; HUANG Daozhong; LI Kaiyan; WANG Zhihui; HONG Kai; WANG Fen; ZANG Qingping

    2007-01-01

    To examine the role of ultrasound in gene delivery in vitro, three cells lines were exposed to the low-frequency ultrasound of varying intensities and for different durations to evaluate their effect on gene transfection and cell viability of the cells. Microbubble (MB), Optison (10%), was also used to observe the role of the microbubbles in gene transfection. The results demonstrated that as the ultrasound intensity and the exposure time increased, the gene transfer rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically, which caused the transfer rate to decrease. The most efficient ultrasound intensity for inducing gene transfer was 1 W/cm2 with duration being 20 s. At the same energy intensity, higher ultrasound intensity could achieve maximal gene transfer rate earlier. Microbubbles could increase ultrasound-induced cell gene transfer rate by about 2 to 3 times mainly at lower energy intensities. Moreover, microbubbles could raise the maximum gene transfer rate mediated by ultrasound. It is concluded that the low-frequency ultrasound can induce cell gene transfer and the cell gene transfer rate and viability are correlated with not only the ultrasound energy intensity but also the ultrasound intensity, the higher ultrasound intensity achieves its maximal transfer rate more quickly and the ultrasound intensity that can induce optimal gene transfer is 1 W/cm2 with duration being 20 s, and microbubbles can significantly increase the maximal gene transfer rate in vitro.

  5. Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng Qiang; Zhen-Fang Du; Min Huang

    2014-01-01

    Objective: To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. Methods: NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle. Results: A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. Conclusions: NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

  6. Horizontal gene transfer from Agrobacterium to plants

    Directory of Open Access Journals (Sweden)

    Tatiana V. Matveeva

    2014-08-01

    Full Text Available Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A.rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named cellular T-DNA (cT-DNA. It represents an imperfect inverted repeat and contains homologues of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14 and an opine synthesis gene (Ngmis. A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologues of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

  7. mdr1启动子调控CD::UPP基因对紫杉醇耐药卵巢癌细胞的杀伤作用%Cell-killing effects of adenovirus-mediated transfer of CD :: UPP gene directed by mdr1 promoter on Taxol-resistant ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    卢实; 蔡俐琼; 王晓翊; 王泽华

    2007-01-01

    目的:探讨腺病毒介导的mdr1启动子调控胞嘧啶脱氨酶::尿嘧啶磷酸核糖转移酶(CD::UPP)融合基因联合5-氟胞嘧啶(5-FC)对紫杉醇耐药卵巢癌细胞的特异性杀伤作用.方法:扩增、纯化含有mdr1-CD::UPP基因的重组腺病毒,转染人卵巢癌紫杉醇耐药细胞株A2780/Taxol和亲本细胞株A2780,RT-PCR检测mdr1和CD::UPP基因的表达水平;之后加入5-FC,MTT法检测细胞抑制情况及旁观者效应,并观察腺病毒转染后裸鼠移植瘤的生长情况.结果:mdr1和CD::UPP基因在A2780/Taxol细胞中可稳定表达,转染后A2780/Taxol组的细胞生长明显低于A2780组;转基因的A2780/Taxol细胞联合5-FC后可通过旁观者效应杀伤周围未转基因的耐药细胞;耐药组移植瘤生长明显受到抑制,肿瘤体积为(569.10±187.93)mm3,对照组肿瘤体积为(2 111.98±230.82)mm3,差异有统计学意义(P<0.01).结论:mdr1启动子可调控CD::UPP基因特异性表达并特异性杀伤紫杉醇耐药卵巢癌细胞.

  8. Progress in gene transfer by germ cells in mammals

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.

  9. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays

    OpenAIRE

    Raut, Shruti; Lei, Pedro; Padmashali, Roshan; Andreadis, Stelios T.

    2010-01-01

    We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decrease...

  10. Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

    DEFF Research Database (Denmark)

    Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas;

    2013-01-01

    BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...

  11. Lentiviral vector gene transfer to porcine airways.

    Science.gov (United States)

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  12. Optical gene transfer by femtosecond laser pulses

    Science.gov (United States)

    Konig, Karsten; Riemann, Iris; Tirlapur, Uday K.

    2003-07-01

    Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany; focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.

  13. Radiopharmaceuticals to monitor the expression of transferred genes in gene transfer therapy

    International Nuclear Information System (INIS)

    The development and application of radiopharmaceuticals has, in many instances, been based on the pharmacological properties of therapeutic agents. The molecular biology-biotechnology revolution has had an important impact on treatment of diseases, in part through the reduced toxicity of 'biologicals', in part because of their specificity for interaction at unique molecular sites and in part because of their selective delivery to the target site. Immunotherapeutic approaches include the use of monoclonal antibodies (MABs), MAB-fragments and chemotactic peptides. Such agents currently form the basis of both diagnostic and immunotherapeutic radiopharmaceuticals. More recently, gene transfer techniques have been advanced to the point that a new molecular approach, gene therapy, has become a reality. Gene therapy offers an opportunity to attack disease at its most fundamental level. The therapeutic mechanism is based on the expression of a specific gene or genes, the product of which will invoke immunological, receptor-based or enzyme-based therapeutic modalities. Several approaches to gene therapy of cancer have been envisioned, the most clinically-advanced concepts involving the introduction of genes that will encode for molecular targets nor normally found in healthy mammalian cells. A number of gene therapy clinical trials are based on the introduction of the Herpes simplex virus type-1 (HSV-1) gene that encodes for viral thymidine kinase (tk+). Once HSV-1 tk+ is expressed in the target (cancer) cell, therapy can be effected by the administration of a highly molecularly-targeted and systemically non-toxic antiviral drug such as ganciclovir. The development of radiodiagnostic imaging in gene therapy will be reviewed, using HSV-1 tk+ and radioiodinated IVFRU as a basis for development of the theme. Molecular targets that could be exploited in gene therapy, other than tk+, will be identified

  14. Radiopharmaceuticals to monitor the expression of transferred genes in gene transfer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, L. I. [University of Alberta, Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

    1997-10-01

    The development and application of radiopharmaceuticals has, in many instances, been based on the pharmacological properties of therapeutic agents. The molecular biology-biotechnology revolution has had an important impact on treatment of diseases, in part through the reduced toxicity of `biologicals`, in part because of their specificity for interaction at unique molecular sites and in part because of their selective delivery to the target site. Immunotherapeutic approaches include the use of monoclonal antibodies (MABs), MAB-fragments and chemotactic peptides. Such agents currently form the basis of both diagnostic and immunotherapeutic radiopharmaceuticals. More recently, gene transfer techniques have been advanced to the point that a new molecular approach, gene therapy, has become a reality. Gene therapy offers an opportunity to attack disease at its most fundamental level. The therapeutic mechanism is based on the expression of a specific gene or genes, the product of which will invoke immunological, receptor-based or enzyme-based therapeutic modalities. Several approaches to gene therapy of cancer have been envisioned, the most clinically-advanced concepts involving the introduction of genes that will encode for molecular targets nor normally found in healthy mammalian cells. A number of gene therapy clinical trials are based on the introduction of the Herpes simplex virus type-1 (HSV-1) gene that encodes for viral thymidine kinase (tk+). Once HSV-1 tk+ is expressed in the target (cancer) cell, therapy can be effected by the administration of a highly molecularly-targeted and systemically non-toxic antiviral drug such as ganciclovir. The development of radiodiagnostic imaging in gene therapy will be reviewed, using HSV-1 tk+ and radioiodinated IVFRU as a basis for development of the theme. Molecular targets that could be exploited in gene therapy, other than tk+, will be identified

  15. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  16. Simultaneous identification of duplications and lateral gene transfers.

    Science.gov (United States)

    Tofigh, Ali; Hallett, Michael; Lagergren, Jens

    2011-01-01

    The incongruency between a gene tree and a corresponding species tree can be attributed to evolutionary events such as gene duplication and gene loss. This paper describes a combinatorial model where so-called DTL-scenarios are used to explain the differences between a gene tree and a corresponding species tree taking into account gene duplications, gene losses, and lateral gene transfers (also known as horizontal gene transfers). The reasonable biological constraint that a lateral gene transfer may only occur between contemporary species leads to the notion of acyclic DTL-scenarios. Parsimony methods are introduced by defining appropriate optimization problems. We show that finding most parsimonious acyclic DTL-scenarios is NP-hard. However, by dropping the condition of acyclicity, the problem becomes tractable, and we provide a dynamic programming algorithm as well as a fixed-parameter tractable algorithm for finding most parsimonious DTL-scenarios.

  17. Pollen irradiation and possible gene transfer in Nicotiana species

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1985-01-01

    , and Petunia parodii with irradiated pollen from N. alata and Petunia hybrida showed no evidence of gene transfer, nor did experiments with irradiated mentor pollen. This indicates that gene transfer with irradiated pollen between non-crossing species or between species giving sterile hybrids is probably...

  18. Identification and Categorization of Horizontally Transferred Genes in Prokaryotic Genomes

    Institute of Scientific and Technical Information of China (English)

    Shuo-Yong SHI; Xiao-Hui CAI; Da-fu DING

    2005-01-01

    Horizontal gene transfer (HGT), a process through which genomes acquire genetic materials from distantly related organisms, is believed to be one of the major forces in prokaryotic genome evolution.However, systematic investigation is still scarce to clarify two basic issues about HGT: (1) what types of genes are transferred; and (2) what influence HGT events over the organization and evolution of biological pathways. Genome-scale investigations of these two issues will advance the systematical understanding of HGT in the context of prokaryotic genome evolution. Having investigated 82 genomes, we constructed an HGT database across broad evolutionary timescales. We identified four function categories containing a high proportion of horizontally transferred genes: cell envelope, energy metabolism, regulatory functions, and transport/binding proteins. Such biased function distribution indicates that HGT is not completely random;instead, it is under high selective pressure, required by function restraints in organisms. Furthermore, we mapped the transferred genes onto the connectivity structure map of organism-specific pathways listed in Kyoto Encyclopedia of Genes and Genomes (KEGG). Our results suggest that recruitment of transferred genes into pathways is also selectively constrained because of the tuned interaction between original pathway members. Pathway organization structures still conserve well through evolution even with the recruitment of horizontally transferred genes. Interestingly, in pathways whose organization were significantly affected by HGT events, the operon-like arrangement of transferred genes was found to be prevalent. Such results suggest that operon plays an essential and directional role in the integration of alien genes into pathways.

  19. Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

    Science.gov (United States)

    Nicholls, Linda I; Ainscow, Edward K; Rutter, Guy A

    2002-03-01

    Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.

  20. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  1. Problems associated with gene transfer and opportunities for microgravity environments

    Energy Technology Data Exchange (ETDEWEB)

    Tennessen, D.J. [Floriculture and Ornamental Horticulture Cornell University, Ithaca, New York14853 (United States)

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of {ital Agrobacterium} mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed. {copyright} {ital 1997 American Institute of Physics.}

  2. Gene transfer for congestive heart failure: update 2013.

    Science.gov (United States)

    Tang, Tong; Hammond, H Kirk

    2013-04-01

    Congestive heart failure is a major cause of morbidity and mortality with increasing social and economic costs. There have been no new high impact therapeutic agents for this devastating disease for more than a decade. However, many pivotal regulators of cardiac function have been identified using cardiac-directed transgene expression and gene deletion in preclinical studies. Some of these increase function of the failing heart. Altering the expression of these pivotal regulators using gene transfer is now either being tested in clinical gene transfer trials, or soon will be. In this review, we summarize recent progress in cardiac gene transfer for clinical congestive heart failure.

  3. Ad-ING4-IRES-IL-24双基因共表达载体的构建及表达%Construction and expression of adenovirus-mediated ING4 and IL-24 co-expression

    Institute of Scientific and Technical Information of China (English)

    盛伟华; 谢宇锋; 缪竞诚; 顾范博; 单云波; 朱晔涵; 陈华昕; 杜贤荣; 杨吉成

    2011-01-01

    GEZ-Term,pcDNA 3.0-IL-24,and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pA dTrack-CMV-ING4-IRES-IL-24,respectively.The pAdTrack-CMV-ING4-IRES-IL-24 transfer vector linearized with Pme Ⅰdigestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination.The resultant pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰdigestion and transfected into the human embryonic kidney 293(QBI-293A)cells by liposome,leading to formation of the recombinant adenoviruses Ad-ING4-IRES-IL-24 co-expressing ING4 and IL-24.Infected the A549 cells by the expanded adenoviruses Ad-ING4-IRES-IL-24,A denovirus-mediated ING4 and IL-24 expression in QBI-293A and A549 cells was examined by RT-PCR and Western blot.The growth-suppressing and apoptosis-inducing effect of Ad-ING4-ERES IL-24 co-expressing ING 4 and IL-24 on A549 human lung carcinoma cells were assessed by MTT assay and FCM,respectively.Results:DNA sequencing showed that the ING4,IRES,and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in QBI-293A and A 549 cells.A denovirus-mediated ING4 and IL-24 co-expression significantly suppressed A549 lung carcinom a cell growth and induced cell apoptosis.The adenoviral vector co-expressing ING 4 and IL-24 mediated by IRES,Ad-ING4-IRES-IL-24,was successfully constructed.Adenovirus-mediated ING 4 and IL-24 co-expression had marked anti-tum or effect in suppressin A549 human lung carcinom a cell growth and inducing cell apoptosis in vitro.Compared with Ad-ING 4 -IRES(growth inhibition ratio at 72h was 42.31%±0.43%,apoptosis rate was 13.30%±1.85%)and AdIRES-IL-24(growth inhibition ratio at 72h was 47.44%±0.39%,apoptosis rate was 12.40%±1.05%),Ad-ING4-IRES IL-24(growth inhibition ratio at 72h is

  4. Nonviral gene transfer strategies to promote bone regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2013-10-01

    Despite the inherent ability of bone to regenerate itself, there are a number of clinical situations in which complete bone regeneration fails to occur. In view of shortcomings of conventional treatment, gene therapy may have a place in cases of critical-size bone loss that cannot be properly treated with current medical or surgical treatment. The purpose of this review is to provide an overview of gene therapy in general, nonviral techniques of gene transfer including physical and chemical methods, RNA-based therapy, therapeutic genes to be transferred for bone regeneration, route of application including ex vivo application, and direct gene therapy approaches to regenerate bone.

  5. Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

    Institute of Scientific and Technical Information of China (English)

    李秀锦; 仲飞; 齐顺章

    2003-01-01

    Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P<0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

  6. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes.

  7. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    Science.gov (United States)

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  8. The power of phylogenetic approaches to detect horizontally transferred genes

    Directory of Open Access Journals (Sweden)

    Gogarten J Peter

    2007-03-01

    Full Text Available Abstract Background Horizontal gene transfer plays an important role in evolution because it sometimes allows recipient lineages to adapt to new ecological niches. High genes transfer frequencies were inferred for prokaryotic and early eukaryotic evolution. Does horizontal gene transfer also impact phylogenetic reconstruction of the evolutionary history of genomes and organisms? The answer to this question depends at least in part on the actual gene transfer frequencies and on the ability to weed out transferred genes from further analyses. Are the detected transfers mainly false positives, or are they the tip of an iceberg of many transfer events most of which go undetected by current methods? Results Phylogenetic detection methods appear to be the method of choice to infer gene transfers, especially for ancient transfers and those followed by orthologous replacement. Here we explore how well some of these methods perform using in silico transfers between the terminal branches of a gamma proteobacterial, genome based phylogeny. For the experiments performed here on average the AU test at a 5% significance level detects 90.3% of the transfers and 91% of the exchanges as significant. Using the Robinson-Foulds distance only 57.7% of the exchanges and 60% of the donations were identified as significant. Analyses using bipartition spectra appeared most successful in our test case. The power of detection was on average 97% using a 70% cut-off and 94.2% with 90% cut-off for identifying conflicting bipartitions, while the rate of false positives was below 4.2% and 2.1% for the two cut-offs, respectively. For all methods the detection rates improved when more intervening branches separated donor and recipient. Conclusion Rates of detected transfers should not be mistaken for the actual transfer rates; most analyses of gene transfers remain anecdotal. The method and significance level to identify potential gene transfer events represent a trade

  9. EFFECTS OF p53 GENE THERAPY COMBINED WITH CYCLOOXYGENASE-2 INHIBITOR ON CYCLOOXYGENASE-2 GENE EXPRESSION AND GROWTH INHIBITION OF HUMAN LUNG CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Zhao-Xia; LU Bin-Bin; WANG Teng; YIN Yong-Mei; DE Wei; SHU Yong-Qian

    2007-01-01

    Background Gene therapy by adenovirus-mediated wild-type p53 gene transfer has been shown to inhibit lung cancer growth in vitro, in animal models, and in human clinical trials. The antitumor effect of selective cyclooxygenase (COX)-2 inhibitors has been demonstrated in preclinical studies. However, no information is available on the effects of p53 gene therapy combined with selective COX-2 inhibitor on COX-2 gene expression and growth inhibition of human lung cancer cells. Methods We evaluated the effects of recombinant adenovirus-p53 (Ad-p53) gene therapy combined with selective COX-2 inhibitor on the proliferation, apoptosis, cell cycle arrest of human lung adenocarcinoma A549 cell line, and the effects of tumor suppressor exogenous wild type p53 on COX-2 gene expression. Results Ad-p53 gene therapy combined with selective COX-2 inhibitor celecoxib shows significant synergistic inhibition effects on the growth of human lung adenocarcinoma A549 cell line. Exogenous p53 gene can suppress COX-2 gene expression. Conclusions Significant synergistic inhibition effects of A549 cell line by the combined Ad-p53 and selective COX-2 inhibitor celecoxib may be achieved by enhancement of growth inhibition, apoptosis induction and suppression of COX-2 gene expression. This study provides first evidence that the administration of p53 gene therapy in combination with COX-2 inhibitors might be a new clinical strategy for the treatment or prevention of NSCLC.

  10. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays.

    Science.gov (United States)

    Raut, Shruti D; Lei, Pedro; Padmashali, Roshan M; Andreadis, Stelios T

    2010-06-01

    We employed fibrin hydrogel as a bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 and 7.5mg/ml. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially-localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine. PMID:20153386

  11. Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

    Directory of Open Access Journals (Sweden)

    Roger Andrew J

    2003-06-01

    Full Text Available Abstract Background Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH, to evaluate and compare the patterns and rates of lateral gene transfer (LGT in prokaryotes and eukaryotes. Results We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists. Conclusion LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

  12. Electroporation-Mediated Gene Transfer Directly to the Swine Heart

    OpenAIRE

    Hargrave, Barbara; Downey, Harre; Strange, Robert; Murray, Len; Cinnamond, Cade; Lundberg, Cathryn; Israel, Annelise; Chen, Yeong-Jer; Marshall, William; Heller, Richard

    2012-01-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using 3 different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the ECG were administered at varying pulse...

  13. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    Science.gov (United States)

    The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium ve...

  14. Gentamicin resistance genes in environmental bacteria: prevalence and transfer

    NARCIS (Netherlands)

    Heuer, H.; Krögerrecklenfort, E.; Wellington, E.M.H.; Egan, S.; Elsas, van J.D.; Overbeek, van L.S.; Collard, J.M.; Guillaume, G.; Karagouni, A.; Nikolakopoulou, D.; Smalla, K.

    2002-01-01

    A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gmr) genes in different non-clinical environments has been performed. We were interested to find out whether Gmr genes described from clinical isolates can be detected in different environmental habitats and

  15. In vivo comparison of transduction efficiency with recombinant adenovirus-mediated p53 in a human colon cancer mouse model by different delivery routes%rAd/p53不同给药途径治疗人类结肠癌荷瘤鼠模型p53导入效率的在体评价

    Institute of Scientific and Technical Information of China (English)

    Qi Xie; Biling Liang; ling Zhang; Qihua Yang; Xiongfei Gu; Jing Xu; Mingwang Chen

    2008-01-01

    Objective: To evaluate transduction efficiency with recombinant adenovirus-mediated p53 (rAd/p53) therapy in a human colon cancer mouse model by intra-tumoral injection and intra-arterial delivery. Methods: The tumor pieces of human colon cancer SW480 were implanted in the livers of 45 nude mice. These mice were administrated with rAd/p53 by intratu-moral injection and intra-arterial delivery. After 24 h, 48 h and 72 h rAd/p53 administration, 5 mice each group were killed with over anesthesia and their livers were removed. P53 expression and apoptosis of tumor and liver were assessed. Results: P53 expression and apoptosis of intratumoral administration group was higher than tail vein group and control group. Apoptosis and p53 expression of livers in three groups had no significant difference. Conclusion: p53 gene transduction efficiency and anticancer effect of tAd/p53 is much better by intra-tumoral injection than intra-arterial delivery.

  16. Amelioration of radiation-induced skin injury by adenovirus-mediated heme oxygenase-1 (HO-1) overexpression in rats

    International Nuclear Information System (INIS)

    Radiation-induced skin injury remains a serious concern for radiation therapy. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant and anti-apoptotic properties. However, the role of HO-1 in radiation-induced skin damage remains unclear. This study aims to elucidate the effects of HO-1 on radiation-induced skin injury in rats. A control adenovirus (Ad-EGFP) and a recombinant adenovirus (Ad-HO1-EGFP) were constructed. Rats were irradiated to the buttock skin with a single dose of 45 Gy followed by a subcutaneous injection of PBS, 5 × 109 genomic copies of Ad-EGFP or Ad-HO1-EGFP (n = 8). After treatment, the skin MDA concentration, SOD activity and apoptosis were measured. The expression of antioxidant and pro-apoptotic genes was determined by RT-PCR and real-time PCR. Skin reactions were measured at regular intervals using the semi-quantitative skin injury score. Subcutaneous injection of Ad-HO1-EGFP infected both epidermal and dermal cells and could spread to the surrounding regions. Radiation exposure upregulated the transcription of the antioxidant enzyme genes, including SOD-1, GPx2 and endogenous HO-1. HO-1 overexpression decreased lipid peroxidation and inhibited the induction of ROS scavenging proteins. Moreover, HO-1 exerted an anti-apoptotic effect by suppressing FAS and FASL expression. Subcutaneous injection of Ad-HO1-EGFP demonstrated significant improvement in radiation-induced skin injury. The present study provides evidences for the protective role of HO-1 in alleviating radiation-induced skin damage in rats, which is helpful for the development of therapy for radiation-induced skin injury

  17. Design of radiopharmaceuticals for monitoring gene transfer therapy

    International Nuclear Information System (INIS)

    The development of radiopharmaceuticals for monitoring gene transfer therapy with emission tomography is expected to lead to improved management of cancer by the year 2010. There are now only a few examples and approaches to the design of radiopharmaceuticals for gene transfer therapy. This paper introduces a novel concept for the monitoring of gene therapy. We present the optimisation of the labelling of recombinant human β-NGF ligands for in vitro studies prior to using 123I for SPET and 124I for PET studies. (author)

  18. Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Walker Thomas

    2009-01-01

    Full Text Available Abstract Background The evolutionary importance of horizontal gene transfer (HGT from Wolbachia endosymbiotic bacteria to their eukaryotic hosts is a topic of considerable interest and debate. Recent transfers of genome fragments from Wolbachia into insect chromosomes have been reported, but it has been argued that these fragments may be on an evolutionary trajectory to degradation and loss. Results We have discovered a case of HGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector. The lower level of sequence identity between Wolbachia and insect, the transcription of all the genes involved, and the fact that we have identified homologs of the two genes in another Aedes species (Ae. mascarensis, suggest that these genes are being expressed after an extended evolutionary period since horizontal transfer, and therefore that the transfer has functional significance. The association of these genes with Wolbachia prophage regions also provides a mechanism for the transfer. Conclusion The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation.

  19. Agrobacterium-mediated gene transfer to Chrysanthemum.

    NARCIS (Netherlands)

    Wordragen, van M.F.

    1991-01-01

    Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is

  20. DNA-mediated gene transfer into ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

  1. Expression of a transferred nuclear gene in a mitochondrial genome

    Directory of Open Access Journals (Sweden)

    Yichun Qiu

    2014-08-01

    Full Text Available Transfer of mitochondrial genes to the nucleus, and subsequent gain of regulatory elements for expression, is an ongoing evolutionary process in plants. Many examples have been characterized, which in some cases have revealed sources of mitochondrial targeting sequences and cis-regulatory elements. In contrast, there have been no reports of a nuclear gene that has undergone intracellular transfer to the mitochondrial genome and become expressed. Here we show that the orf164 gene in the mitochondrial genome of several Brassicaceae species, including Arabidopsis, is derived from the nuclear ARF17 gene that codes for an auxin responsive protein and is present across flowering plants. Orf164 corresponds to a portion of ARF17, and the nucleotide and amino acid sequences are 79% and 81% identical, respectively. Orf164 is transcribed in several organ types of Arabidopsis thaliana, as detected by RT-PCR. In addition, orf164 is transcribed in five other Brassicaceae within the tribes Camelineae, Erysimeae and Cardamineae, but the gene is not present in Brassica or Raphanus. This study shows that nuclear genes can be transferred to the mitochondrial genome and become expressed, providing a new perspective on the movement of genes between the genomes of subcellular compartments.

  2. Agrobacterium-mediated gene transfer to Chrysanthemum.

    OpenAIRE

    Wordragen, van, M.F.

    1991-01-01

    Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is a random process in which two complete genomes are mixed and the desired phenotype has to be regained by repeated back crossing with the cultivated parent line. Despite these differences, both pro...

  3. Important aspects of placental-specific gene transfer.

    Science.gov (United States)

    Kaufman, Melissa R; Albers, Renee E; Keoni, Chanel; Kulkarni-Datar, Kashmira; Natale, David R; Brown, Thomas L

    2014-10-15

    The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

  4. The interconnection between biofilm formation and horizontal gene transfer.

    Science.gov (United States)

    Madsen, Jonas Stenløkke; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2012-07-01

    Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.

  5. Gene therapy of cancer and development of therapeutic target gene

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang Min; Kwon, Hee Chung

    1998-04-01

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

  6. Gene therapy of cancer and development of therapeutic target gene

    International Nuclear Information System (INIS)

    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene

  7. Gene transfer strategies in animal transgenesis.

    Science.gov (United States)

    Montoliu, Lluís

    2002-01-01

    Position effects in animal transgenesis have prevented the reproducible success and limited the initial expectations of this technique in many biotechnological projects. Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression domain. An expression domain is thought to contain all regulatory elements that are needed to specifically control the expression of a given gene in time and space. The lack of profound knowledge on the chromatin structure of expression domains of biotechnological interest, such as mammary gland-specific genes, explains why most standard expression vectors have failed to drive high-level, position-independent, and copy-number-dependent expression of transgenes in a reproducible manner. In contrast, the application of artificial chromosome-type constructs to animal transgenesis usually ensures optimal expression levels. YACs, BACs, and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant key regulatory sequences, previously unknown, that are characteristic for each expression domain. These elements contribute to insulating the artificial chromosome-type constructs from chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes.

  8. Experiments on Gene Transferring to Primary Hematopoietic Cells by Liposome

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by Xgal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33±2. 68) % in human and about (16. 28±2.95) % in mouse without G418 selection. After G418 selection, the blue cell rate was (46. 06±3.47)%in human and (43. 45±4. 1) % in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia.

  9. Detecting Horizontal Gene Transfer between Closely Related Taxa.

    Directory of Open Access Journals (Sweden)

    Orit Adato

    2015-10-01

    Full Text Available Horizontal gene transfer (HGT, the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM. Using CRM, the algorithm assigns a confidence score based on "unusual" sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain.

  10. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

    Directory of Open Access Journals (Sweden)

    Nico Lachmann

    2013-03-01

    Full Text Available Gene transfer of drug resistance (CTX-R genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C, gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.

  11. Effects of ionizing radiation on DNA-mediated gene transfer

    International Nuclear Information System (INIS)

    The process of DNA-mediated gene transfer is a powerful genetic tool that involves the cellular uptake, genomic integration and expression of exogenous DNA sequences. This process can also be used to examine the effects of radiation at the molecular level. There have been a few reported describing the enhancement of the gene transfer process by a number of DNA damaging agents. The agents tested included UV light, x-rays and accelerated argon particles. One hypothesis to explain this phenomenon is that these DNA damaging agents themselves, or subsequent DNA repair processes, introduce strand breaks into the cellular DNA of recipient cells. These DNA breaks then serve as possible sites of integration for the exogenous DNA sequences. The authors are continuing these studies by determining what effect neutrons have on the transfection of DNA. The gene transfer system we plan to employ involves the transfection of the chimeric plasmid pSV2-GPT into recipient hamster cell lines. This plasmid contains the Escherichia coli ecogpt gene, which codes for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT), along with simian virus 40 (SV40) sequences which allow for expression of the bacterial gene in mammalian cells

  12. Horizontal gene transfer in the evolution of photosynthetic eukaryotes

    Institute of Scientific and Technical Information of China (English)

    Jinling HUANG; Jipei YUE

    2013-01-01

    Horizontal gene transfer (HGT) may not only create genome mosaicism,but also introduce evolutionary novelties to recipient organisms.HGT in plastid genomes,though relatively rare,still exists.HGT-derived genes are particularly common in unicellular photosynthetic eukaryotes and they also occur in multicellular plants.In particular,ancient HGT events occurring during the early evolution of primary photosynthetic eukaryotes were probably frequent.There is clear evidence that anciently acquired genes played an important role in the establishment of primary plastids and in the transition of plants from aquatic to terrestrial environments.Although algal genes have often been used to infer historical plastids in plastid-lacking eukaryotes,reliable approaches are needed to distinguish endosymbionts-derived genes from those independently acquired from preferential feeding or other activities.

  13. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    Science.gov (United States)

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation. PMID:25733873

  14. Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaobo Chen; Yongxiang Li; Weiping Dong; Yang Jiao; Jianming Tan

    2007-01-01

    Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively.The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index (SI) was calculated. Glucose-stimulated insulin release was usedto assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index(SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets(P < 0.05).Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

  15. Regulatory and Ethical Issues for Phase I In Utero Gene Transfer Studies

    OpenAIRE

    Strong, Carson

    2011-01-01

    Clinical gene transfer research has involved adult and child subjects, and it is expected that gene transfer in fetal subjects will occur in the future. Some genetic diseases have serious adverse effects on the fetus before birth, and there is hope that prenatal gene therapy could prevent such disease progression. Research in animal models of prenatal gene transfer is actively being pursued. The prospect of human phase I in utero gene transfer studies raises important regulatory and ethical i...

  16. Immunotherapy of Malignancy by in vivo Gene Transfer into Tumors

    Science.gov (United States)

    Plautz, Gregory E.; Yang, Zhi-Yong; Wu, Bei-Yue; Gao, Xiang; Huang, Leaf; Nabel, Gary J.

    1993-05-01

    The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2K^s gene within the CT26 mouse colon adenocarcinoma (H-2K^d) or the MCA 106 fibrosarcoma (H-2K^b) induced a cytotoxic T-cell response to H-2K^s and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

  17. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    International Nuclear Information System (INIS)

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (NeoR) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact NeoR gene integration and expressed high levels of neomycin phosphotransferase activity. The NeoR gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for β- and γ-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the β and γ chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors (α and β) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs

  18. Direct Gene Transfer into Rabbit Peripheral Nerve in vivo

    Institute of Scientific and Technical Information of China (English)

    张世强; 张经歧; 张英泽; 刘玲

    2001-01-01

    Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of anastomoses under the condition that perfect nerve anastomoses are ensured. PCMVβ plasmid containing cytomegalovirus promoter (CMV promoter) and Escherichia coli (E.coli) β-Galactosidase (β-Gal) structural gene (lacZ gene) was conducted. A soaked medical 8-0nylon suture was used to perform epineurial repair of rabbit sciatic nerve. In the control group a suture soaked in sucrose PBS was used, while in the experimental group a suture soaked in PCMVβ plasmid solution was applied. The sites of anastomoses of nerves by stages were taken out, and β-Gal histochemical staining was performed and β-Gal enzyme activity was assayed with 5-bromo-4-chloro-3-indolyl-β-D-galactoside. Results showed that the sites of anastomoses of nerves were taken out 2 days, 7 days, 14 days and 30 days respectively after the operation. The β-Gal histochemical stains at the sites of anastomoses showed no indigo positive cells at different stages in the control group, whereas displayed indigo positive cells in the experimental group. In the control group, no β-Gal enzyme activity was detected at different stages after operation, but in the experimental group, β-Gal enzyme activity could be detected from the 3rd day to the 30th day after operation. It was concluded that by using exogenous gene suture, exogenous gene could be transferred to the sites of peripheral nerve and expressed the exogenous gene expression products with bioactivity, which provided the feasibility of using gene therapy to accelerate the recovery of nerve function.

  19. Characterization of an ancient lepidopteran lateral gene transfer.

    Directory of Open Access Journals (Sweden)

    David Wheeler

    Full Text Available Bacteria to eukaryote lateral gene transfers (LGT are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31 from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65-145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea. Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.

  20. Electroporation-mediated gene transfer directly to the swine heart.

    Science.gov (United States)

    Hargrave, B; Downey, H; Strange, R; Murray, L; Cinnamond, C; Lundberg, C; Israel, A; Chen, Y-J; Marshall, W; Heller, R

    2013-02-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were killed 48 h after injection and electroporation and gene expression was determined. Results were compared with sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared with injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

  1. Adenovirus serotype 5 hexon mediates liver gene transfer.

    Science.gov (United States)

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  2. Gene Transfer To Intact Mesenteric Arteries by Electroporation

    OpenAIRE

    Martin, Jason B.; Young, Jennifer L.; Benoit, Joseph N.; Dean, David A.

    2000-01-01

    The purpose of the present study was to develop a rapid, reproducible method of non-viral gene transfer to the intact vasculature. Male Sprague-Dawley rats were anesthetized, a midline abdominal incision was made and segmental branches of the superior mesenteric artery were dissected free of surrounding mesentery. A specially designed electroporation probe was placed around the neurovascular bundle and the electroporation chamber filled with a solution containing the firefly luciferase expres...

  3. Detection of homologous horizontal gene transfer in SNP data

    Energy Technology Data Exchange (ETDEWEB)

    2012-07-23

    We study the detection of mutations, sequencing errors, and homologous horizontal gene transfers (HGT) in a set of closely related microbial genomes. We base the model on single nucleotide polymorphisms (SNP's) and break the genomes into blocks to handle the rearrangement problem. Then we apply a synamic programming algorithm to model whether changes within each block are likely a result of mutations, sequencing errors, or HGT.

  4. Selective Gene Transfer to the Retina Using Intravitreal Ultrasound Irradiation

    Directory of Open Access Journals (Sweden)

    Shozo Sonoda

    2012-01-01

    Full Text Available This paper aims to evaluate the efficacy of intravitreal ultrasound (US irradiation for green fluorescent protein (GFP plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50 μL and bubble liposomes (BLs; 50 μL was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72 h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM of GFP-positive cells (32±4.9; n=7; P<0.01 . No GFP-positive cells were observed in the control eyes (n=7. Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value.

  5. The interconnection between biofilm formation and horizontal gene transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenløkke; Burmølle, Mette; Hansen, Lars H.;

    2012-01-01

    Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their beli......Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because...... of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states....... Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids...

  6. Stable oncogenic transformation induced by microcell-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    吕有勇; Donald G.Blair

    1995-01-01

    Oncogenes have been identified using DNA-mediated transfection, but the size of the transferable and unrearranged DNA, gene rearrangement and amplification which occur during the transfection process limit the use of the techniques. We have evaluated microcell-mediated gene transfer techniques for the transfer and analysis of dominant oncogenes. MNNG-HOS, a transformed human cell line which contained the met oncogene mapping to human chromosome 7 was infected with retroviruses carrying drug resistance markers and used to optimize microcell preparation and transfer. Stable and drug-resistant hybrids containing single human chromosomes as well as the foci of the transformed cells containing the activated met oncogene and intact hitman chromosomes were obtained. Hybridization analysis with probes (i.e. collA2, pJ3.11) mapping up to 1 Mb away from met shows that the cells from the individual focr contain different amounts of apparently unrearranged human DNA associated with the oncogene, and the microcell-g

  7. A rice Stowaway MITE for gene transfer in yeast.

    Directory of Open Access Journals (Sweden)

    Isam Fattash

    Full Text Available Miniature inverted repeat transposable elements (MITEs lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo on a construct bearing dual reporter genes (gfp and neo was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes.

  8. Dynamic monitoring of horizontal gene transfer in soil

    Science.gov (United States)

    Cheng, H. Y.; Masiello, C. A.; Silberg, J. J.; Bennett, G. N.

    2015-12-01

    Soil microbial gene expression underlies microbial behaviors (phenotypes) central to many aspects of C, N, and H2O cycling. However, continuous monitoring of microbial gene expression in soils is challenging because genetically-encoded reporter proteins widely used in the lab are difficult to deploy in soil matrices: for example, green fluorescent protein cannot be easily visualized in soils, even in the lab. To address this problem we have developed a reporter protein that releases small volatile gases. Here, we applied this gas reporter in a proof-of-concept soil experiment, monitoring horizontal gene transfer, a microbial activity that alters microbial genotypes and phenotypes. Horizontal gene transfer is central to bacterial evolution and adaptation and is relevant to problems such as the spread of antibiotic resistance, increasing metal tolerance in superfund sites, and bioremediation capability of bacterial consortia. This process is likely to be impacted by a number of matrix properties not well-represented in the petri dish, such as microscale variations in water, nutrients, and O2, making petri-dish experiments a poor proxy for environmental processes. We built a conjugation system using synthetic biology to demonstrate the use of gas-reporting biosensors in safe, lab-based biogeochemistry experiments, and here we report the use of these sensors to monitor horizontal gene transfer in soils. Our system is based on the F-plasmid conjugation in Escherichia coli. We have found that the gas signal reports on the number of cells that acquire F-plasmids (transconjugants) in a loamy Alfisol collected from Kellogg Biological Station. We will report how a gas signal generated by transconjugants varies with the number of F-plasmid donor and acceptor cells seeded in a soil, soil moisture, and soil O2 levels.

  9. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    Science.gov (United States)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  10. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory.

    Science.gov (United States)

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G; Van Leeuwen, Thomas

    2016-01-01

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants. PMID:27307274

  11. Evidence of horizontal gene transfer between obligate leaf nodule symbionts.

    Science.gov (United States)

    Pinto-Carbó, Marta; Sieber, Simon; Dessein, Steven; Wicker, Thomas; Verstraete, Brecht; Gademann, Karl; Eberl, Leo; Carlier, Aurelien

    2016-09-01

    Bacteria of the genus Burkholderia establish an obligate symbiosis with plant species of the Rubiaceae and Primulaceae families. The bacteria, housed within the leaves, are transmitted hereditarily and have not yet been cultured. We have sequenced and compared the genomes of eight bacterial leaf nodule symbionts of the Rubiaceae plant family. All of the genomes exhibit features consistent with genome erosion. Genes potentially involved in the biosynthesis of kirkamide, an insecticidal C7N aminocyclitol, are conserved in most Rubiaceae symbionts. However, some have partially lost the kirkamide pathway due to genome erosion and are unable to synthesize the compound. Kirkamide synthesis is therefore not responsible for the obligate nature of the symbiosis. More importantly, we find evidence of intra-clade horizontal gene transfer (HGT) events affecting genes of the secondary metabolism. This indicates that substantial gene flow can occur at the early stages following host restriction in leaf nodule symbioses. We propose that host-switching events and plasmid conjugative transfers could have promoted these HGTs. This genomic analysis of leaf nodule symbionts gives, for the first time, new insights in the genome evolution of obligate symbionts in their early stages of the association with plants. PMID:26978165

  12. Targeted adenovirus mediated inhibition of NF-kappa B-dependent inflammatory gene expression in endothelial cells in vitro and in vivo

    NARCIS (Netherlands)

    Kuldo, J. M.; Asgeirsdottir, S. A.; Zwiers, P. J.; Bellu, A. R.; Rots, M. G.; Schalk, J. A. C.; Ogawara, K. I.; Trautwein, C.; Banas, B.; Haisma, H. J.; Molema, G.; Kamps, J. A. A. M.

    2013-01-01

    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor kappa B signal transduction could silence the proinflammatory activation status of en

  13. Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea

    DEFF Research Database (Denmark)

    Frigaard, Niels-Ulrik; Martinez, Asuncion; Mincer, Tracy J;

    2006-01-01

    Planktonic Bacteria, Archaea and Eukarya reside and compete in the ocean's photic zone under the pervasive influence of light. Bacteria in this environment were recently shown to contain photoproteins called proteorhodopsins, thought to contribute to cellular energy metabolism by catalysing light......-driven proton translocation across the cell membrane. So far, proteorhodopsin genes have been well documented only in proteobacteria and a few other bacterial groups. Here we report the presence and distribution of proteorhodopsin genes in Archaea affiliated with the order Thermoplasmatales, in the ocean......'s upper water column. The genomic context and phylogenetic relationships of the archaeal and proteobacterial proteorhodopsins indicate its probable lateral transfer between planktonic Bacteria and Archaea. About 10% of the euryarchaeotes in the photic zone contained the proteorhodopsin gene adjacent...

  14. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    Science.gov (United States)

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR. PMID:23912835

  15. Detecting lateral gene transfers by statistical reconciliation of phylogenetic forests

    Directory of Open Access Journals (Sweden)

    Daubin Vincent

    2010-06-01

    Full Text Available Abstract Background To understand the evolutionary role of Lateral Gene Transfer (LGT, accurate methods are needed to identify transferred genes and infer their timing of acquisition. Phylogenetic methods are particularly promising for this purpose, but the reconciliation of a gene tree with a reference (species tree is computationally hard. In addition, the application of these methods to real data raises the problem of sorting out real and artifactual phylogenetic conflict. Results We present Prunier, a new method for phylogenetic detection of LGT based on the search for a maximum statistical agreement forest (MSAF between a gene tree and a reference tree. The program is flexible as it can use any definition of "agreement" among trees. We evaluate the performance of Prunier and two other programs (EEEP and RIATA-HGT for their ability to detect transferred genes in realistic simulations where gene trees are reconstructed from sequences. Prunier proposes a single scenario that compares to the other methods in terms of sensitivity, but shows higher specificity. We show that LGT scenarios carry a strong signal about the position of the root of the species tree and could be used to identify the direction of evolutionary time on the species tree. We use Prunier on a biological dataset of 23 universal proteins and discuss their suitability for inferring the tree of life. Conclusions The ability of Prunier to take into account branch support in the process of reconciliation allows a gain in complexity, in comparison to EEEP, and in accuracy in comparison to RIATA-HGT. Prunier's greedy algorithm proposes a single scenario of LGT for a gene family, but its quality always compares to the best solutions provided by the other algorithms. When the root position is uncertain in the species tree, Prunier is able to infer a scenario per root at a limited additional computational cost and can easily run on large datasets. Prunier is implemented in C++, using

  16. Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

    Science.gov (United States)

    Wu, Bo; Novelli, Jacopo; Jiang, Daojun; Dailey, Harry A; Landmann, Frédéric; Ford, Louise; Taylor, Mark J; Carlow, Clotilde K S; Kumar, Sanjay; Foster, Jeremy M; Slatko, Barton E

    2013-05-01

    Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.

  17. Adenovirus-mediated expression of pig α(1, 3) galactosyltransferase reconstructs Gal α(1, 3) Gal epitope on the surface of human tumor cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Gal α(1,3)Gal(gal epitope)is a carbohydrate epitope and synthesized in large amount by α(1,3)galactosyltransferase [α(1,3)GT] enzyme on the cells of lower mammalian animals such as pigs and mice.Human has no gal epitope due to the inactivation of α(1,3)GT gene but produces a large amount of antibodies(anti-Gal)which recognize Gal α(1,3)Gal structures specifically.In this study,a replicationdeficient recombinant adenoviral vector Ad5sGT containing pig α(1,3)GT cDNA was constructed and characterized.Adenoviral vector-mediated transfer of pig α(1,3)GT gene into human tumor cells such as malignant melanoma A375,stomach cancer SGC-7901,and lung cancer SPC-A-1 was reported for the first time.Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis,although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction.Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I(UEA I)lectins,Vicia villosa agglutinin(VVA),Arachis hypogaea agglutinin(PNA),and Glycine max agglutinin(SBA)to different degrees.In addition,no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

  18. In vivo Cytokine Gene Transfer by Gene Gun Reduces Tumor Growth in Mice

    Science.gov (United States)

    Sun, Wenn H.; Burkholder, Joseph K.; Sun, Jian; Culp, Jerilyn; Turner, Joel; Lu, Xing G.; Pugh, Thomas D.; Ershler, William B.; Yang, Ning-Sun

    1995-03-01

    Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor α and interferon γ genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon γ genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon γ were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

  19. Differences in lateral gene transfer in hypersaline versus thermal environments

    Directory of Open Access Journals (Sweden)

    House Christopher H

    2011-07-01

    Full Text Available Abstract Background The role of lateral gene transfer (LGT in the evolution of microorganisms is only beginning to be understood. While most LGT events occur between closely related individuals, inter-phylum and inter-domain LGT events are not uncommon. These distant transfer events offer potentially greater fitness advantages and it is for this reason that these "long distance" LGT events may have significantly impacted the evolution of microbes. One mechanism driving distant LGT events is microbial transformation. Theoretically, transformative events can occur between any two species provided that the DNA of one enters the habitat of the other. Two categories of microorganisms that are well-known for LGT are the thermophiles and halophiles. Results We identified potential inter-class LGT events into both a thermophilic class of Archaea (Thermoprotei and a halophilic class of Archaea (Halobacteria. We then categorized these LGT genes as originating in thermophiles and halophiles respectively. While more than 68% of transfer events into Thermoprotei taxa originated in other thermophiles, less than 11% of transfer events into Halobacteria taxa originated in other halophiles. Conclusions Our results suggest that there is a fundamental difference between LGT in thermophiles and halophiles. We theorize that the difference lies in the different natures of the environments. While DNA degrades rapidly in thermal environments due to temperature-driven denaturization, hypersaline environments are adept at preserving DNA. Furthermore, most hypersaline environments, as topographical minima, are natural collectors of cellular debris. Thus halophiles would in theory be exposed to a greater diversity and quantity of extracellular DNA than thermophiles.

  20. NAC1, a POZ/BTB protein present in the adult mammalian brain, triggers apoptosis after adenovirus-mediated overexpression in PC-12 cells.

    Science.gov (United States)

    Korutla, Laxminarayana; Neustadter, Jason H; Fournier, Keith M; Mackler, Scott A

    2003-05-01

    POZ/BTB proteins influence cellular development and in some examples act as oncoproteins. However, several POZ/BTB transcription factors have been found in terminally differentiated neurons, where their functions remain unknown. One example is NAC1, a constitutively-expressed protein that can regulate behaviors associated with cocaine use. The present study represents an initial attempt to understand the actions of NAC1 within neurons by using adenoviral-mediated gene transfer into differentiated PC-12 cells. Cell survival in PC-12 cells overexpressing NAC1 was greatly reduced compared with cells infected by a control Ad-GFP. The morphological appearance of the dying cells was consistent with programmed cell death. Fragmentation of genomic DNA occurred in PC-12 cells infected with adenoviruses encoding NAC1 but not control viruses. NAC1 over expression was followed by the down regulation of the anti-apoptotic proteins Bcl-2 and Bcl-2-xl. Concurrently, levels of the pro-apoptotic proteins Bax and p53 increased following NAC1 overexpression. These observations suggest that NAC1expression in PC-12 cells induces apoptosis by altering the expression of these upstream mediators of the execution phase of programmed cell death. These findings raise the possibility that aberrantly regulated NAC1 expression in the mammalian brain may contribute to programmed cell death.

  1. Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer.

    OpenAIRE

    Evans, G A; Ingraham, H A; Lewis, K; Cunningham, K; Seki, T.; Moriuchi, T; Chang, H. C.; Silver, J; Hyman, R

    1984-01-01

    We isolated a gene encoding the Thy-1.2 glycoprotein from a recombinant library constructed from BALB/c mouse DNA. To evaluate the expression of this cloned gene in different genomic environments, we introduced it into cell lines derived from fibroblast, lymphoid, and neuronal tissues by DNA-mediated gene transfer. When integrated into the genome of mouse L cells, cell-surface Thy-1 can be detected with anti-Thy-1 monoclonal antibodies. These L-cell lines contain between two and four copies o...

  2. Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.

  3. Genome-wide experimental determination of barriers to horizontal gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward; Sorek, Rotem; Zhu, Yiwen; Creevey, Christopher J.; Francino, M. Pilar; Bork, Peer; Rubin, Edward M.

    2007-09-24

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into E. coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Their toxicity to the host inhibited transfer regardless of the species of origin and our data suggest that increased gene dosage and associated increased expression is a predominant cause for transfer failure. While these experimental studies examined transfer solely into E. coli, a computational analysis of gene transfer rates across available bacterial and archaeal genomes indicates that the barriers observed in our study are general across the tree of life.

  4. GENE TRANSFER IN TOBACCO MITOCHONDRIA IN VITRO AND IN VIVO

    Directory of Open Access Journals (Sweden)

    Katyshev A.I.

    2012-08-01

    Full Text Available Earlier, we had showed that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility of genes transfer in tobacco mitochondria in vitro and in vivo. Whereas homologous recombination is a rare occasion in higher plant nuclei, recombination between the large direct repeats in plant mitochondrial genome generates its multipartite structure. Following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by mitochondrial DNA fragments, we showed the homologous recombination of imported DNA with the resident DNA and the integration of the reporter gene. The recombination yielded an insertion of a continuous exogenous DNA fragment including the gfp sequence and at least the 0.5 kb of the flanking sequence on each side. Using of transfection constructs carrying multiple sequences homologous to mitochondrial DNA could be suitable for insertion of a target gene into any region of the mitochondrial genome, which turns this approach to be of a general and methodical importance. Usually mitochondrial reactive oxygen species (ROS level is under strict control of the antioxidant system including the Mn-containing superoxide dismutase (MnSOD. MnSOD is presented in multiple forms encoded by several genes in plants. Possibly, this enzyme, beside its catalytic function, fulfills as well some unknown biochemical functions. Thus, one of maize SOD enzymes (SOD3.4 could bind with mitochondrial DNA. Another SOD form (SOD3.1 is located in close proximity to mitochondrial respiratory complexes, where ROS are generated. To study possible physiological functions of this enzyme, we cloned the maize SOD3.1 gene. Compared to the SOD3.4, this enzyme didn't demonstrate DNA-binding activity. At the same time, SOD3.1 didn't show non-specific DNA-hydrolyzing activity as Cu/ZnSOD does. It means that this enzyme might have some DNA protective function. We made NtPcob-sod3.1-IGR

  5. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    Science.gov (United States)

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  6. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

    Directory of Open Access Journals (Sweden)

    Anton V Borovjagin

    Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

  7. Chromosomal nif Genes Transfer by Conjugation in Nitrogen Fixing Azotobacter chroococcum to Lactobacillus plantarium

    OpenAIRE

    Adel Kamal Khider; Aras Muhammad Khidher

    2011-01-01

    To determine the possibility of transferring chromosomal nitrogen fixation genes (nif genes) from Azotobacter chroococcum to Lactobacillus planetarium, a total of 72 Azotobacter chroococcum isolated from Erbil governorate, Iraq were culturally, morphologically and biochemically characterized. Genes for atmospheric nitrogen fixation, located on the chromosome of Azotobacter chroococcum isolates were transferred by conjugation process to a recipient Lactobacillus plantarium isolated from Erbil ...

  8. NANOPARTICLE AS A NEW GENE TRANSFERRING VECTOR IN SPECIFIC EXPRESSION GENE

    Institute of Scientific and Technical Information of China (English)

    管珩; 李拥军; 郑曰宏; 刘昌伟; 杨菁; 宋存先; 王彭延; 赵三妹; 王宗立; 佘铭鹏

    2002-01-01

    Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) beating antisense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP - 1(200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.

  9. Extensive intra-kingdom horizontal gene transfer converging on a fungal fructose transporter gene.

    Directory of Open Access Journals (Sweden)

    Marco A Coelho

    2013-06-01

    Full Text Available Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1 with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed.

  10. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    Science.gov (United States)

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  11. Statistical Mechanics of Horizontal Gene Transfer in Evolutionary Ecology

    Science.gov (United States)

    Chia, Nicholas; Goldenfeld, Nigel

    2011-04-01

    The biological world, especially its majority microbial component, is strongly interacting and may be dominated by collective effects. In this review, we provide a brief introduction for statistical physicists of the way in which living cells communicate genetically through transferred genes, as well as the ways in which they can reorganize their genomes in response to environmental pressure. We discuss how genome evolution can be thought of as related to the physical phenomenon of annealing, and describe the sense in which genomes can be said to exhibit an analogue of information entropy. As a direct application of these ideas, we analyze the variation with ocean depth of transposons in marine microbial genomes, predicting trends that are consistent with recent observations using metagenomic surveys.

  12. Immortalized neural progenitor cells for CNS gene transfer and repair.

    Science.gov (United States)

    Martínez-Serrano, A; Björklund, A

    1997-11-01

    Immortalized multipotent neural stem and progenitor cells have emerged as a highly convenient source of tissue for genetic manipulation and ex vivo gene transfer to the CNS. Recent studies show that these cells, which can be maintained and genetically transduced as cell lines in culture, can survive, integrate and differentiate into both neurons and glia after transplantation to the intact or damaged brain. Progenitors engineered to secrete trophic factors, or to produce neurotransmitter-related or metabolic enzymes can be made to repopulate diseased or injured brain areas, thus providing a new potential therapeutic tool for the blockade of neurodegenerative processes and reversal of behavioural deficits in animal models of neurodegenerative diseases. With further technical improvements, the use of immortalized neural progenitors may bring us closer to the challenging goal of targeted and effective CNS repair.

  13. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    Science.gov (United States)

    Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  14. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    Directory of Open Access Journals (Sweden)

    Yingmei Peng

    Full Text Available Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC while the other as bacteria-type pepcase (BTPC because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

  15. Passive Immunization against HIV/AIDS by Antibody Gene Transfer

    Directory of Open Access Journals (Sweden)

    Lili Yang

    2014-01-01

    Full Text Available Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP, which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics.

  16. Center for fetal monkey gene transfer for heart, lung, and blood diseases: an NHLBI resource for the gene therapy community.

    Science.gov (United States)

    Tarantal, Alice F; Skarlatos, Sonia I

    2012-11-01

    The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; "proof-of-principle"; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field.

  17. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    Directory of Open Access Journals (Sweden)

    Daniell Henry

    2011-06-01

    Full Text Available Abstract Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun" delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI methods. Results Plasmid DNA carrying the firefly luciferase (LUC reporter gene under the control of the human Cytomegalovirus (CMV promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter using different DNA Loading Ratios (DLRs, and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50 at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results

  18. Efficiency of adenoviral vector mediated CTLA4Ig gene delivery into mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    邓宇斌; 郭小荑; 原清涛; 李树浓

    2003-01-01

    Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. Methods We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. Results Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P<0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation. Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.

  19. Evolutionary advantage conferred by an eukaryote-to-eukaryote gene transfer event in wine yeasts

    OpenAIRE

    Marsit, Souhir; Mena, Adriana; Bigey, Frederic; Sauvage, Francois Xavier; Couloux, Arnaud; Guy, Julie; Legras, Jean Luc; Barrio, Eladio; Dequin, Sylvie

    2015-01-01

    Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding ol...

  20. The Use of Viral Vectors in Gene Transfer Therapy

    OpenAIRE

    Dziaková, A.; Valenčáková, A.; Hatalová, E.; J. Kalinová

    2016-01-01

    Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including canc...

  1. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  2. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene.

    Science.gov (United States)

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-07-27

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese's complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes.

  3. Differential gene transfers and gene duplications in primary and secondary endosymbioses

    Directory of Open Access Journals (Sweden)

    McFadden Geoffrey I

    2006-04-01

    Full Text Available Abstract Background Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, groEL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of groEL homologues. Results Our analyses indicate that while two distinct groEL genes were introduced into eukaryotes by a progenitor of plastids, these particular homologues have not been maintained in all evolutionary lineages. This is of significant interest, because two chaperone proteins always co-occur in oxygenic photosynthetic organisms. We infer strikingly different lineage specific processes of evolution involving deletion, duplication and targeting of groEL proteins. Conclusion The requirement of two groEL homologues for chaperon function in phototrophs has provided a constraint that has shaped convergent evolutionary scenarios in divergent evolutionary lineages. GroEL provides a general evolutionary model for studying gene transfers and convergent evolutionary processes among eukaryotic lineages.

  4. Estimating the extent of horizontal gene transfer in metagenomic sequences

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2008-03-01

    Full Text Available Abstract Background Although the extent of horizontal gene transfer (HGT in complete genomes has been widely studied, its influence in the evolution of natural communities of prokaryotes remains unknown. The availability of metagenomic sequences allows us to address the study of global patterns of prokaryotic evolution in samples from natural communities. However, the methods that have been commonly used for the study of HGT are not suitable for metagenomic samples. Therefore it is important to develop new methods or to adapt existing ones to be used with metagenomic sequences. Results We have created two different methods that are suitable for the study of HGT in metagenomic samples. The methods are based on phylogenetic and DNA compositional approaches, and have allowed us to assess the extent of possible HGT events in metagenomes for the first time. The methods are shown to be compatible and quite precise, although they probably underestimate the number of possible events. Our results show that the phylogenetic method detects HGT in between 0.8% and 1.5% of the sequences, while DNA compositional methods identify putative HGT in between 2% and 8% of the sequences. These ranges are very similar to these found in complete genomes by related approaches. Both methods act with a different sensitivity since they probably target HGT events of different ages: the compositional method mostly identifies recent transfers, while the phylogenetic is more suitable for the detections of older events. Nevertheless, the study of the number of HGT events in metagenomic sequences from different communities shows a consistent trend for both methods: the lower amount is found for the sequences of the Sargasso Sea metagenome, while the higher quantity is found in the whale fall metagenome from the bottom of the ocean. The significance of these observations is discussed. Conclusion The computational approaches that are used to find possible HGT events in complete

  5. The Use of Viral Vectors in Gene Transfer Therapy

    Directory of Open Access Journals (Sweden)

    A. Dziaková

    2016-05-01

    Full Text Available Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA, single-stranded DNA (ssDNA, plasmid DNA and antisense oligodeoxynucleotides (ASON. The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%, retroviruses (344 clinical studies; 20.5%, unenveloped/plasmid DNA (304 clinical studies, 17.7%, adeno-associated viruses (75 clinical studies; 4.5% and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.

  6. A first glimpse into the pattern and scale of gene transfer in the Apicomplexa

    DEFF Research Database (Denmark)

    Huang, J.L.; Mullapudi, N.; Sicheritz-Pontén, Thomas;

    2004-01-01

    with a phylogenomic approach to detect potential gene transfers in four apicomplexan genomes. We have detected genes of algal nuclear, chloroplast (cyanobacterial) and proteobacterial origin. Plant-like genes were detected in species not currently harbouring a plastid (e.g. Cryptosporidium parvum) and putatively...

  7. Assessment and Improvement of Gene Transfer into Human Hematopoietic Stem Cells

    NARCIS (Netherlands)

    D.A. Breems (Dimitri)

    1997-01-01

    textabstractThe application of somatic gene transfer as a potential treatment in human disease has progressed from speculation to reality in a short time [4,20,21,84,85,87,105,117,174]. In May 1989 the first clinical marker gene protocol took place [145], followed by the first gene therapy protocol

  8. Prostate Specific Antigen Promoter-Driven Adenovirus-Mediated Expression of Both ODC and AdoMetDC Antisenses Inhibit Prostate Cancer Growth

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Hui Xiong; Yi-lin Hong; Chun-hua Zhang; Chang-chun Liu

    2010-01-01

    Objective:To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase(ODC)and S-adenosylmethionine decarboxylase(AdoMetDC)antisenses specifically in prostate cancer cells,and evaluate its inhibitory effect on prostate cancer in vivo.Methods:Fragments of ODC and AdoMetDC genes were generated by PCR,cloned into the pPGL-PSES,and then recombined with pAdEasy-1 vectors in AdEasy-1 cells.Ad-PSES-ODC-AdoMetDCas virus was produced in HEK293 cells.Following transfection with Ad-PSES-ODC-AdoMetDCas,the levels of ODC or AdoMetDC were determined by RT-PCR and western blot assays.The effect of Ad-PSES-ODC-AdoMetDCas treatment on tumor formation and growth was evaluated in xenograft models of prostate cancers in vivo.Results:The plasmid pAdEasy-PSES-ODC-AdoMetDCas was successfully constructed and the recombinant Ad-PSES-ODC-AdoMetDCas adenovirus was produced.Transfection with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the expression of ODC and AdoMetDC genes specifically in prostate DU145cells,but not H1299,HT29 and HepG2 cancer cells,and disrupted the ability of DU145 cells to form solid prostate cancer in vivo.Intratumoral treatment with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the growth of engrafted prostate tumors in vivo.Conclusion:The recombinant Ad-PSES-ODC-AdoMetDCas adenovirus specifically reduces the expression of both ODC and AdoMetDC genes in prostate cells and may be used for treatment of prostate cancers at the clinic.

  9. Prostate Specific Antigen Promoter-Driven Adenovirus-Mediated Expression of Both ODC and AdoMetDC Antisenses Inhibit Prostate Cancer Growth

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Hui Xiong; Yi-lin Hong; Chun-hua Zhang; Chang-chun Liu

    2011-01-01

    Objective: To generate recombinant adenovirus that could simultaneously express ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase(AdoMetDC) antisenses specifically in prostate cancer cells,and evaluate its inhibitory effect on prostate cancer in vivo.Methods: Fragments of ODC and AdoMetDC genes were generated by PCR,cloned into the pPGL-PSES,and then recombined with pAdEasy-1 vectors in AdEasy-1 cells.Ad-PSES-ODC-AdoMetDCas virus was produced in HEK293 cells.Following transfection with Ad-PSES-ODC-AdoMetDCas,the levels of ODC or AdoMetDC were determined by RT-PCR and western blot assays.The effect of Ad-PSES-ODC-AdoMetDCas treatment on tumor formation and growth was evaluated in xenograft models of prostate cancers in vivo.Results: The plasmid pAdEasy-PSES-ODC-AdoMetDCas was successfully constructed and the recombinant Ad-PSES-ODC-AdoMetDCas adenovirus was produced.Transfection with Ad-PSES-ODC-AdoMetDCasadenovirus significantly inhibited the expression of ODC and AdoMetDC genes specifically in prostate DU145 cells,but not H1299,HT29 and HepG2 cancer cells,and disrupted the ability of DU145 cells to form solid prostate cancer in vivo.Intratumoral treatment with Ad-PSES-ODC-AdoMetDCas adenovirus significantly inhibited the growth of engrafted prostate tumors in vivo.both ODC and AdoMetDC genes in prostate cells and may be used for treatment of prostate cancers at the clinic.

  10. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  11. Leu452His mutation in lipoprotein lipase gene transfer associated with hypertriglyceridemia in mice in vivo.

    Directory of Open Access Journals (Sweden)

    Kaiyue Sun

    Full Text Available Mutated mouse lipoprotein lipase (LPL containing a leucine (L to histidine (H substitution at position 452 was transferred into mouse liver by hydrodynamics-based gene delivery (HD. Mutated-LPL (MLPL gene transfer significantly increased the concentrations of plasma MLPL and triglyceride (TG but significantly decreased the activity of plasma LPL. Moreover, the gene transfer caused adiposis hepatica and significantly increased TG content in mouse liver. To understand the effects of MLPL gene transfer on energy metabolism, we investigated the expression of key functional genes related to energy metabolism in the liver, epididymal fat, and leg muscles. The mRNA contents of hormone-sensitive lipase (HSL, adipose triglyceride lipase (ATGL, fatty acid-binding protein (FABP, and uncoupling protein (UCP were found to be significantly reduced. Furthermore, we investigated the mechanism by which MLPL gene transfer affected fat deposition in the liver, fat tissue, and muscle. The gene expression and protein levels of forkhead Box O3 (FOXO3, AMP-activated protein kinase (AMPK, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α were found to be remarkably decreased in the liver, fat and muscle. These results suggest that the Leu452His mutation caused LPL dysfunction and gene transfer of MLPL in vivo produced resistance to the AMPK/PGC-1α signaling pathway in mice.

  12. Horizontal gene transfer and the evolution of transcriptionalregulation in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P.

    2007-12-20

    Background: Most bacterial genes were acquired by horizontalgene transfer from other bacteria instead of being inherited bycontinuous vertical descent from an ancient ancestor}. To understand howthe regulation of these {acquired} genes evolved, we examined theevolutionary histories of transcription factors and of regulatoryinteractions from the model bacterium Escherichia coli K12. Results:Although most transcription factors have paralogs, these usually arose byhorizontal gene transfer rather than by duplication within the E. colilineage, as previously believed. In general, most neighbor regulators --regulators that are adjacent to genes that they regulate -- were acquiredby horizontal gene transfer, while most global regulators evolvedvertically within the gamma-Proteobacteria. Neighbor regulators wereoften acquired together with the adjacent operon that they regulate, sothe proximity might be maintained by repeated transfers (like "selfishoperons"). Many of the as-yet-uncharacterized (putative) regulators havealso been acquired together with adjacent genes, so we predict that theseare neighbor regulators as well. When we analyzed the histories ofregulatory interactions, we found that the evolution of regulation byduplication was rare, and surprisingly, many of the regulatoryinteractions that are shared between paralogs result from convergentevolution. Another surprise was that horizontally transferred genes aremore likely than other genes to be regulated by multiple regulators, andmost of this complex regulation probably evolved after the transfer.Conclusions: Our results highlight the rapid evolution of niche-specificgene regulation in bacteria.

  13. Conjugal gene transfer between bacteria in soil and rhizosphere.

    NARCIS (Netherlands)

    Smit, E.

    1994-01-01

    The extent of possible conjugal transfer of recombinant DNA present in genetically engineered microorganisms (GEMs) was studied. Occurrence of transfer of recombinant DNA is only one of the concerns regarding the use of GEMs (Chapter 2). Other potential hazards preventing the application of GEMs for

  14. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus.

    Science.gov (United States)

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-08-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton-virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  15. Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

    Science.gov (United States)

    Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

    2011-09-01

    In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

  16. Transfer of engineered genes from crop to wild plants

    DEFF Research Database (Denmark)

    Bagger Jørgensen, Rikke; Hauser, T.P.; Mikkelsen, T.R.;

    1996-01-01

    The escape of engineered genes - genes inserted using recombinant DNA techniques - from cultivated plants to wild or weedy relatives has raised concern about possible risks to the environment or to health. The media have added considerably to public concern by suggesting that such gene escape...

  17. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits

    OpenAIRE

    Li, Wenju; Wei, Haifeng; Xia, Chunmei; Zhu, Xiaomeng; Hou, Guozhu; Xu, Feng; Xinghua SONG; Zhan, Yulin

    2015-01-01

    Purpose: Transferring the bone morphogenetic protein 2 (BMP-2) genes into the tissues or cells can improve the bone healing of the fracture has been widely accepted. We evaluated the efficiency of using gene gun to transfer the BMP-2 gene thereby affected the healing of a fractured bone. Methods: The vector coding for BMP-2 was constructed by a non-replicating encephalo-myocarditis virus (ECMV)-based vector. The segmental bone defect (1.5 cm) model was created by a wire-saw at the middle part...

  18. Overexpression of the promyelocytic leukemia gene suppresses growth of human bladder cancer cells by inducing G1 cell cycle arrest and apoptosis

    Institute of Scientific and Technical Information of China (English)

    HE Dalin 贺大林; NAN Xunyi 南勋义; Chang Kun-Song; WANG Yafeng 王亚峰; Chung Leland W.K.

    2003-01-01

    Objectives To examine the anti-oncogenic effects of promyelocytic leukemia (PML) on bladder cancer and to explore its molecular mechanisms of growth suppression.Methods Wild-type PML was transfected into bladder cancer cells (5637 cell) and expressed in a replication-deficient adenovirus-mediated gene delivery system and introduced into human bladder cancer cells (5637 cell) in vitro and in vivo. The effect and mechanisms of the PML gene in cell growth, clonogenicity, and tumorigenicity of bladder cancer cells were studied using in vitro and in vivo growth assays, soft agar colony-forming assay, cell cycle analysis, apoptosis assay and in vivo tumorigenicity assay.Results Overexpression of PML in 5637 cells significantly reduced their growth rate and clonogenicity on soft agar. PML suppressed bladder cancer cell growth by inducing G1 cell cycle arrest and apoptosis. Adenovirus-mediated PML (Ad-PML) significantly suppressed the tumorigenicity and growth of bladder cancer cells. Intratumoral injection of Ad-PML into tumors induced by 5637 cells dramatically suppressed their growth. Conclusions The results indicated that overexpression of PML protein may promote efficient growth inhibition of human bladder cancer cells by inducing G1 cell cycle arrest and apoptosis, and adenovirus-mediated PML (Ad-PML) expression efficiently suppresses human bladder cancer growth.

  19. Intrapleural 'outside-in' gene therapy: therapeutics for organs of the chest via gene transfer to the pleura.

    Science.gov (United States)

    Heguy, Adriana; Crystal, Ronald G

    2005-10-01

    The pleural space is an attractive site for using viral vectors to deliver gene products to the lung parenchyma, other thoracic structures and the systemic circulation. The advantages of intrapleural gene transfer using viral vectors include: (i) easy accessibility; (ii) large surface area; (iii) ability to provide high concentrations of secreted gene products to chest structures; (iv) low risk of detrimental effects of possible vector-induced inflammation compared with intravascular delivery; and (v) because it is local, lower vector doses can be used to deliver therapeutic genes to thoracic structures than less efficient systemic routes. Examples of pleural gene transfer include the use of adenovirus vectors to treat mesothelioma by transiently expressing genes that encode toxic proteins, immunomodulatory molecules or anti-angiogenesis factors. Intrapleural delivery of adeno-associated viral vectors represents an efficient strategy to treat alpha1-antitrypsin (alpha1AT) deficiency, achieving high lung and systemic therapeutic levels of alpha1AT. Intrapleural delivery of gene transfer vectors holds promise for the treatment of diseases requiring transient, localized gene expression, as well as sustained expression of genes to correct hereditary disorders requiring localized or systemic expression of the therapeutic protein. PMID:16248279

  20. Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

    International Nuclear Information System (INIS)

    The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.)

  1. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model.

    Science.gov (United States)

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4(+)CD25(+) regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4(+) or CD8(+) cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3(DTR) mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  2. ADENOVIRUS-MEDIATED EXPRESSION OF PEX, A NONCATALYTIC FRAGMENT OF MATRIX METALLOPROTEINASE-2, AND IT'S INHIBITION ON ANGIOGENESIS AND TUMOR GROWTH

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To develop an adenovirus system to deliver biologically active peptides or proteins such as angiogenesis inhibitors in vivo for the treatment of cancer. Methods: DNA recombination techniques were employed to construct adenovirus shuttle vector, in which angiogenesis inhibitor was put downstream of rat growth hormone signal peptide, and the C-terminal was the myc-epitope 10-amino-acid peptide for the following up of the protein. Adenovirus was made using the bacteria recombination method. We tested this system using an angiogenesis inhibitor chick MMP-2 C-terminal hemopexin-like fragment (PEX) in Sarcoma 180 (S-180) bearing Kunming mice. The anti-angiogenic effect was performed by chick chorioallantoic membrane assay. Results: PEX was readily secreted outside human stomach carcinoma BGC823 cells as demonstrated by immunofluorescent staining and western blot infected by adenovirus with rat growth hormone signal peptide (E-T-rGH-PEX). However, without signal peptide (E-T-PEX), PEX was expressed and localized in the cytoplasm of the infected cells, and formed large aggregates, which suggested that PEX was insoluble. The adenovirus E-T-rGH-PEX could inhibit angiogenesis, while E-T-rGH-PEX not. The adenoviruses of E-T-rGH-PEX inhibited the growth of S-180 tumor significantly compared with the empty virus control group E-T (P=0.026) and without signal peptide group E-T-PEX (P=0.006) respectively, while E-T-PEX had little effect. Conclusion: These results suggest that this adenoviral system is likely to be used in the gene therapy of cancer to deliver angiogenesis inhibitors.

  3. Gene transfer system for the phytopathogenic fungus Ustilago maydis.

    OpenAIRE

    Wang, J.; Holden, D. W.; Leong, S A

    1988-01-01

    A selectable marker for transformation was constructed by transcriptional fusion of a Ustilago maydis heat shock gene promoter with the hygromycin B phosphotransferase gene of Escherichia coli. U. maydis was transformed to hygromycin B resistance by polyethylene glycol-induced fusion of spheroplasts following exposure to plasmid DNA that carried the marker gene. Transformation frequencies of 50 and 1000 transformants per microgram of DNA per 2 x 10(7) spheroplasts were obtained for circular a...

  4. Regulated expression of foreign genes in vivo after germline transfer.

    OpenAIRE

    Passman, R S; Fishman, G I

    1994-01-01

    Tight transcriptional control of foreign genes introduced into the germline of transgenic mice would be of great experimental value in studies of gene function. To develop a system in which the spatial and temporal expression of candidate genes implicated in cardiac development or function could be tightly controlled in vivo, we have generated transgenic mice expressing a tetracycline-controlled transactivator (tTA) under the control of a rat alpha myosin heavy chain promoter (MHC alpha-tTA m...

  5. DNA-mediated gene transfer in plant protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    U, Zang Kual; Riu, Key Zung; So, In Sup; Hong, Kyung Ae [Cheju National University, Cheju (Korea, Republic of)

    1994-12-31

    The neomycin phosphotransferase II gene(NPT-II) was introduced into geranium (Pelargonium zonale hybrids) protoplasts by using PEG or electroporation method. The presence of the introduced DNA in the protoplasts and the expressions of the gene in the transformed cells were examined. The presence of the NPT-II DNA in the protoplasts were detected by polymerase chain reaction. The expressions of NPT-II gene in the transformed cells were confirmed by the NPT-II assay. (author)

  6. The impact of non-electrical factors on electrical gene transfer

    OpenAIRE

    Hu, Jiemiao; CUTRERA, JEFFRY; LI, SHULIN

    2014-01-01

    Electrical pulses directly and effectively boost both in vitro and in vivo gene transfer, but this process is greatly affected by non-electrical factors that exist during electroporation. These factors include, but are not limited to, the types of cells or tissues used, the property of DNA, DNA formulation, and the expressed protein. In this mini-review, we only describe and discuss a summary of DNA properties and selected DNA formulations on gene transfer via electroporation. The properties ...

  7. Evolutionary change and phylogenetic relationships in light of horizontal gene transfer

    Indian Academy of Sciences (India)

    Luis Boto

    2015-06-01

    Horizontal gene transfer has, over the past 25 years, become a part of evolutionary thinking. In the present paper I discuss horizontal gene transfer (HGT) in relation to contingency, natural selection, evolutionary change speed and the Tree-of-Life endeavour, with the aim of contributing to the understanding of the role of HGT in evolutionary processes. In addition, the challenges that HGT imposes on the current view of evolution are emphasized.

  8. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    OpenAIRE

    Shih Ping Yao; Ho Pei-Yu; Huang Hsiao-I; Bolen James; Brown Lucy; Hsiao Chin-Ton; Lo Hsin-Lung; Lai Chao-Kuen; Chen Chi-Dar; Wu Ming-Che; Liu Yi-Hsin; Jiang MeiSheng; Qian Jin; Chang Keejong; Yao Chen-Wen

    2002-01-01

    Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal ...

  9. Feline Immunodeficiency Virus as a Gene Transfer Vector in the Rat Nucleus Tractus Solitarii

    OpenAIRE

    Lin, L. H.; Langasek, J. E.; Talman, L. S.; Taktakishvili, O. M.; Talman, W. T.

    2009-01-01

    Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effect...

  10. Active Methyl Cycle and Transfer Related Gene Expression in Response to Drought Stress in Rice Leaves

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-li; ZHOU Jian; HAN Zhuo; SHANG Qi; WANG Ze-gang; GU Xiao-hui; GE Cai-lin

    2012-01-01

    Three rice varieties,Zhonghan 3,Shanyou 63 and Aizizhan,were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress.The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution.The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress.Furthermore,drought stress could induce the expression of a large number of methyltransferase genes,especially the transcription of Rubisco protein methylation related genes,which are beneficial for prevention of Rubisco protein oxidation and degradation,and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes.This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.

  11. In silico Analysis of the Potential Infection Mechanisms of Magnaporthe grisea from Horizontal Gene Transfer Hypothesis

    Institute of Scientific and Technical Information of China (English)

    Chunyang Li; Ying Wang; Hao Peng; Hejiao Bian; Mingwei Min; Longfei Chen; Qian Liu; Jinku Bao

    2009-01-01

    Horizontal gene transfer(HGT)has long been considered as a principal force for an organism to gain novel genes in genome evolution. Homology search, phylogenetic analysis and nucleotide composition analysis are three major objective approaches to arguably determine the occurrence and directionality of HGT. Here, 21 genes that possess the potential to horizontal transfer were acquired from the whole genome of Magnaporthe grisea according to annotation, among which three can-didate genes(corresponding protein accession numbers are EAA55123, EAA47200 and EAA52136)were selected for further analysis. According to BLAST homology results, we subsequently conducted phylogenetic analysis of the three candidate HGT genes. Moreover, nucleotide composition analysis was conducted to further validate these HGTs. In addition, the functions of the three candidate genes were searched in COG database. Consequently, we conclude that the gene encoding protein EAA55123 is transferred from Clostridium perfringens. Another HGT event is between EAA52136 and a certain metazoan's corresponding gene, but the direction remains uncertain. Yet, EAA47200 is not a transferred gene.

  12. Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria.

    Science.gov (United States)

    Demanèche, Sandrine; Monier, Jean-Michel; Dugat-Bony, Eric; Simonet, Pascal

    2011-10-01

    The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes. PMID:21564143

  13. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    Science.gov (United States)

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  14. Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes.

    Science.gov (United States)

    Yabuki, Akinori; Toyofuku, Takashi; Takishita, Kiyotaka

    2014-07-01

    Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.

  15. ENHANCED ANTITUMOR EFFECTS OF SUICIDE GENE THERAPY BY SIMULTANEOUS TRANSFER OF GMCSF GENE IN LEUKEMIA-BEARING MICE

    Institute of Scientific and Technical Information of China (English)

    Ju Dianwen; Cao Xuetao; Yu Yizhi; Tao Qun; Wang Baomei; Wan Tao

    1998-01-01

    In the present report, antitumor effect of combined transfer of suicide gene and cytokine gene was studied.Adenovirus engineered to express E. Coli. Cytosine deaminase (AdCD) and/or adenovirus engineered toexpress murine granulocyte-macrophage colonystimulating factor (AdGMCSF) were used for the treatment of leukemia-bearing mice. The mice were inoculated s.c. With FBL-3 erythroleukemia cells and 3days later received intratumoral injection of AdCD in the presence or absence of AdGMCSF followed by intraperitoneal 5-fluorocytosine (5FC) treatment. The results demonstrated that mice received combined therapy of AdCD/5FC and AdGMCSF developed tumors most slowly and survived much longer when compared with mice treated with AdCD/5FC alone, AdGMCSF alone, AdlacZ/5FC or PBS. Combined transfer of CD gene and GM-CSF gene achieved higher specific CTL activity than control therapies. Pathological examination illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration in mice after combined therapy. The results demonstrated that combined transfer of suicide gene and cytokine gene could synergistically inhibit the growth of leukemia in mice and induce antitumor immunity of the host. The combination therapy might be a potential approach for cancer gene therapy.

  16. Field Supervisory Test of DREB-Transgenic Populus: Salt Tolerance, Long-Term Gene Stability and Horizontal Gene Transfer

    Directory of Open Access Journals (Sweden)

    Nan Lu

    2014-05-01

    Full Text Available Improving saline resistance may be useful for reducing environmental susceptibility and improving yields in poplar plantations. However, the instability of genetically engineered traits and gene transfer reduce their usefulness and commercial value. To investigate whether the foreign gene is still present in the genome of receptor plants after seven years (i.e., long-term foreign gene stability and gene transfer, we randomly analyzed ten field-grown transgenic hybrid Populus ((Populus tomentosa × Populus bolleana × P. tomentosa carrying the DREB1 gene from Atriplex hortensis. The results of PCR and tissue culture experiments showed that AhDREB1 was present in the transgenic trees and was still expressed. However, the transcriptional expression level had decreased compared with that four years earlier. The PCR results also indicated no foreign gene in the genomic DNA of microorganisms in the soil near the transgenic poplars, indicating that no significant gene transfer had occurred from the transgenic poplars to the microorganisms at seven years after planting.

  17. The use of carboxymethylcellulose gel to increase non-viral gene transfer in mouse airways.

    Science.gov (United States)

    Griesenbach, Uta; Meng, Cuixiang; Farley, Raymond; Wasowicz, Marguerite Y; Munkonge, Felix M; Chan, Mario; Stoneham, Charlotte; Sumner-Jones, Stephanie G; Pringle, Ian A; Gill, Deborah R; Hyde, Stephen C; Stevenson, Barbara; Holder, Emma; Ban, Hiroshi; Hasegawa, Mamoru; Cheng, Seng H; Scheule, Ronald K; Sinn, Patrick L; McCray, Paul B; Alton, Eric W F W

    2010-03-01

    We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25-1.5%) was mixed with complexes of the cationic lipid GL67A and plasmids encoding luciferase and perfused onto the nasal epithelium of mice. Survival after perfusion with 1% CMC or 1% MC was 90 and 100%, respectively. In contrast 1.5% CMC was uniformly lethal likely due to the viscous solution blocking the airways. Perfusion with 0.5% CMC containing lipid/DNA complexes reproducibly increased gene expression by approximately 3-fold (n=16, p<0.05). Given this benefit, likely related to increased duration of contact, we also assessed the effect of prolonging contact time of the liposome/DNA complexes by delivering our standard 80 microg DNA dose over either approximately 22 or 60 min of perfusion. This independently increased gene transfer by 6-fold (n=8, p<0.05) and could be further enhanced by the addition of 0.5% CMC, leading to an overall 25-fold enhancement (n=8, p<0.001) in gene expression. As a result of these interventions CFTR transgene mRNA transgene levels were increased several logs above background. Interestingly, this did not lead to correction of the ion transport defects in the nasal epithelium of cystic fibrosis mice nor for immunohistochemical quantification of CFTR expression. To assess if 0.5% CMC also increased gene transfer in the mouse lung, we used whole body nebulisation chambers. CMC was nebulised for 1h immediately before, or simultaneously with GL67A/pCIKLux. The former did not increase gene transfer, whereas co-administration significantly increased gene transfer by 4-fold (p<0.0001, n=18). This study suggests that contact time of non-viral gene transfer agents is a key factor for gene delivery, and suggests two methods which may be translatable for use in man. PMID:20022367

  18. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence.

    Science.gov (United States)

    Romero, Miguel; Cerritos, R; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  19. Kinetics of conjugative gene transfer on surfaces in granular porous media

    Science.gov (United States)

    Massoudieh, A.; Crain, C.; Lambertini, E.; Nelson, K. E.; Barkouki, T.; L'Amoreaux, P.; Loge, F. J.; Ginn, T. R.

    2010-03-01

    The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998).

  20. The standard lateral gene transfer model is statistically consistent for pectinate four-taxon trees

    DEFF Research Database (Denmark)

    Sand, Andreas; Steel, Mike

    2013-01-01

    Evolutionary events such as incomplete lineage sorting and lateral gene transfers constitute major problems for inferring species trees from gene trees, as they can sometimes lead to gene trees which conflict with the underlying species tree. One particularly simple and efficient way to infer...... species trees from gene trees under such conditions is to combine three-taxon analyses for several genes using a majority vote approach. For incomplete lineage sorting this method is known to be statistically consistent; however, for lateral gene transfers it was recently shown that a zone...... of inconsistency exists for a specific four-taxon tree topology, and it was posed as an open question whether inconsistencies could exist for other four-taxon tree topologies? In this letter we analyze all remaining four-taxon topologies and show that no other inconsistencies exist....

  1. Efficient retrovirus-mediated transfer of cell-cycle control genes to transformed cells

    Directory of Open Access Journals (Sweden)

    B.E. Strauss

    1999-07-01

    Full Text Available The use of gene therapy continues to be a promising, yet elusive, alternative for the treatment of cancer. The origins of cancer must be well understood so that the therapeutic gene can be chosen with the highest chance of successful tumor regression. The gene delivery system must be tailored for optimum transfer of the therapeutic gene to the target tissue. In order to accomplish this, we study models of G1 cell-cycle control in both normal and transformed cells in order to understand the reasons for uncontrolled cellular proliferation. We then use this information to choose the gene to be delivered to the cells. We have chosen to study p16, p21, p53 and pRb gene transfer using the pCL-retrovirus. Described here are some general concepts and specific results of our work that indicate continued hope for the development of genetically based cancer treatments.

  2. Limitations of the murine nose in the development of nonviral airway gene transfer.

    Science.gov (United States)

    Griesenbach, Uta; Sumner-Jones, Stephanie G; Holder, Emma; Munkonge, Felix M; Wodehouse, Theresa; Smith, Stephen N; Wasowicz, Marguerite Y; Pringle, Ian; Casamayor, Isabel; Chan, Mario; Coles, Rebecca; Cornish, Nikki; Dewar, Ann; Doherty, Ann; Farley, Raymond; Green, Anne-Marie; Jones, Bryony L; Larsen, Mia D B; Lawton, Anna E; Manvell, Michelle; Painter, Hazel; Singh, Charanjit; Somerton, Lucinda; Stevenson, Barbara; Varathalingam, Anusha; Siegel, Craig; Scheule, Ronald K; Cheng, Seng H; Davies, Jane C; Porteous, David J; Gill, Deborah R; Boyd, A Christopher; Hyde, Steve C; Alton, Eric W F W

    2010-07-01

    A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials. PMID:19648474

  3. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    Science.gov (United States)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  4. Gene Loss and Horizontal Gene Transfer Contributed to the Genome Evolution of the Extreme Acidophile "Ferrovum".

    Science.gov (United States)

    Ullrich, Sophie R; González, Carolina; Poehlein, Anja; Tischler, Judith S; Daniel, Rolf; Schlömann, Michael; Holmes, David S; Mühling, Martin

    2016-01-01

    Acid mine drainage (AMD), associated with active and abandoned mining sites, is a habitat for acidophilic microorganisms that gain energy from the oxidation of reduced sulfur compounds and ferrous iron and that thrive at pH below 4. Members of the recently proposed genus "Ferrovum" are the first acidophilic iron oxidizers to be described within the Betaproteobacteria. Although they have been detected as typical community members in AMD habitats worldwide, knowledge of their phylogenetic and metabolic diversity is scarce. Genomics approaches appear to be most promising in addressing this lacuna since isolation and cultivation of "Ferrovum" has proven to be extremely difficult and has so far only been successful for the designated type strain "Ferrovum myxofaciens" P3G. In this study, the genomes of two novel strains of "Ferrovum" (PN-J185 and Z-31) derived from water samples of a mine water treatment plant were sequenced. These genomes were compared with those of "Ferrovum" sp. JA12 that also originated from the mine water treatment plant, and of the type strain (P3G). Phylogenomic scrutiny suggests that the four strains represent three "Ferrovum" species that cluster in two groups (1 and 2). Comprehensive analysis of their predicted metabolic pathways revealed that these groups harbor characteristic metabolic profiles, notably with respect to motility, chemotaxis, nitrogen metabolism, biofilm formation and their potential strategies to cope with the acidic environment. For example, while the "F. myxofaciens" strains (group 1) appear to be motile and diazotrophic, the non-motile group 2 strains have the predicted potential to use a greater variety of fixed nitrogen sources. Furthermore, analysis of their genome synteny provides first insights into their genome evolution, suggesting that horizontal gene transfer and genome reduction in the group 2 strains by loss of genes encoding complete metabolic pathways or physiological features contributed to the observed

  5. Gene loss and horizontal gene transfer contributed to the genome evolution of the extreme acidophile Ferrovum

    Directory of Open Access Journals (Sweden)

    Sophie Roxana Ullrich

    2016-05-01

    Full Text Available Acid mine drainage (AMD, associated with active and abandoned mining sites, is a habitat for acidophilic microorganisms that gain energy from the oxidation of reduced sulfur compounds and ferrous iron and that thrive at pH below 4. Members of the recently proposed genus Ferrovum are the first acidophilic iron oxidizers to be described within the Betaproteobacteria. Although they have been detected as typical community members in AMD habitats worldwide, knowledge of their phylogenetic and metabolic diversity is scarce. Genomics approaches appear to be most promising in addressing this lacuna since isolation and cultivation of Ferrovum has proven to be extremely difficult and has so far only been successful for the designated type strain Ferrovum myxofaciens P3G. In this study, the genomes of two novel strains of Ferrovum (PN-J185 and Z-31 derived from water samples of a mine water treatment plant were sequenced. These genomes were compared with those of Ferrovum sp. JA12 that also originated from the mine water treatment plant, and of the type strain (P3G. Phylogenomic scrutiny suggests that the four strains represent three Ferrovum species that cluster in two groups (1 and 2. Comprehensive analysis of their predicted metabolic pathways revealed that these groups harbor characteristic metabolic profiles, notably with respect to motility, chemotaxis, nitrogen metabolism, biofilm formation and their potential strategies to cope with the acidic environment. For example, while the F. myxofaciens strains (group 1 appear to be motile and diazotrophic, the non-motile group 2 strains have the predicted potential to use a greater variety of fixed nitrogen sources. Furthermore, analysis of their genome synteny provides first insights into their genome evolution, suggesting that horizontal gene transfer and genome reduction in the group 2 strains by loss of genes encoding complete metabolic pathways or physiological features contributed to the observed

  6. Synthetic gene transfer vectors II: back to the future.

    Science.gov (United States)

    Behr, Jean-Paul

    2012-07-17

    The discovery of RNA interference has given a new lease on life to both the chemistry of oligonucleotides and chemical approaches for the intracellular delivery of nucleic acids. In particular, delivery of siRNA, whether in vitro for screening and target validation purposes or in humans as a new class of drugs, may revolutionize our approach to therapy. Their impact could equal that of the bioproduction and various uses of monoclonal antibodies today. Unfortunately, global pharmaceutical companies again seem to be waiting to buy the next Genentech or Genzyme of gene silencing rather than investing research and development into this promising area of research. Gene silencing encounters barriers similar to gene addition and hence may benefit from the extra decade of experience brought by gene therapy. "Chemical" transfection of cells in culture has become routine, and this Account discusses some of the reasons this success has not extended to nonviral gene therapy trials, most of which do not progress beyond the phase 2 stage. The author also discusses a (much debated) mechanism of nucleic acid cell entry and subsequent release of the polycationic particles into the cytoplasm. Both topics should be useful to those interested in delivery of siRNA. The move from gene therapy toward siRNA as an oligonucleotide-based therapy strategy provides a much wider range of druggable targets. Even though these molecules are a hundredfold smaller than a gene, they are delivered via similar cellular mechanisms. Their complexes with cationic polymers are less stable than those with a higher number of phosphate groups, which may be compensated by siRNA concatemerization or by chemical conjugation with the cationic carrier. Thus chemistry is again desperately needed. PMID:22311735

  7. The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

    Directory of Open Access Journals (Sweden)

    Bradley L. Bearson

    2014-02-01

    Full Text Available Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the U.S. during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness

  8. Cellular automata-based artificial life system of horizontal gene transfer

    Directory of Open Access Journals (Sweden)

    Ji-xin Liu

    2016-02-01

    Full Text Available Mutation and natural selection is the core of Darwin's idea about evolution. Many algorithms and models are based on this idea. However, in the evolution of prokaryotes, more and more researches have indicated that horizontal gene transfer (HGT would be much more important and universal than the authors had imagined. Owing to this mechanism, the prokaryotes not only become adaptable in nearly any environment on Earth, but also form a global genetic bank and a super communication network with all the genes of the prokaryotic world. Under this background, they present a novel cellular automata model general gene transfer to simulate and study the vertical gene transfer and HGT in the prokaryotes. At the same time, they use Schrodinger's life theory to formulate some evaluation indices and to discuss the intelligence and cognition of prokaryotes which is derived from HGT.

  9. Gene transfer during surgical procedures with molecular surgical suture

    Directory of Open Access Journals (Sweden)

    Dan Huang

    2009-06-01

    Full Text Available Over the last decades, there has been an explosion of interest in plasmid DNA for gene therapy with reports of their efficacy in the fight against cancer, vascular diseases, and inherited diseases caused by specific gene defects (Srivastava, 2003. DNA plasmids present several advantages over the use of recombinant viruses concerning their production and safety issues. Plasmid DNA vectors can be constructed easily and economically, and they are free of size constraints imposed by viral packaging, obviating the need for an infectious vector and lessening the likelihood of toxicity and immunogenicity (Davis, 1993. Plasmids have a relative low cost, long shelf life and allow repetitive administration of the therapeutic gene without generating an immune response against the delivery vector (Donnelly, 2003. Finally, plasmids can be injected directly into tissues, such as heart (Sarkar, 2002, muscle (Neumeister, 2001, Dan, 2000 and tumors (De Marco, 2003, Sasaki, 2002.

  10. Antitumor effects of interleukin-18 gene-modified hepatocyte cell line on implanted liver carcinoma

    Institute of Scientific and Technical Information of China (English)

    冷建杭; 张立煌; 姚航平; 曹雪涛

    2003-01-01

    Objective To investigate the antitumor effects of intrasplenically transplanted interleukin-18 (IL-18) gene-modified hepatocytes on murine implanted liver carcinoma.Methods Embryonic murine hepatocyte cell line (BNL-CL2) was transfected with a recombinant adenovirus encoding IL-18 and used as delivery cells for IL-18 gene transfer. Two cell lines, BNL-LacZ and BNL-CL2, were used as controls. One week after intrasplenic injection of C26 cells (colon carcinoma line), tumor-bearing syngeneic mice underwent the intrasplenic transplantation of IL-18 gene-modified hepatocyte cell line and were divided into treatment group (BNL IL-18) and control groups (BNL-LacZ and BNL-CL2 ). Two weeks later, the serum levels of IL-18, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in the implanted liver carcinoma-bearing mice were assayed, the cytotoxicity of murine splenic cytotoxic T-lymphocytes (CTLs) was measured, and the morphology of the hepatic tumors was studied to evaluate the antitumor effects of the approach. Results In the treatment group, the serum levels of IL-18, IFN-γ, TNF-α and NO increased significantly. The splenic CTL activity increased markedly (P<0.01) , accompanied by a substantial decrease in tumor volume and the percentage of tumor area and prolonged survival of liver carcinomo-being mice.Conclusions In vivo IL-18 expression by ex vivo manipulated cells with IL-18 recombinant adenovirus is able to exert potent antitumor effects by inducing a predominantly T-cell-helper type 1 (Th1) immune response. Intrasplenic transplantation of adenovirus-mediated IL-18 gene-modified hepatocytes could be used as a targeting treatment for implanted liver carcinoma.

  11. Smelt was the likely beneficiary of an antifreeze gene laterally transferred between fishes

    Directory of Open Access Journals (Sweden)

    Graham Laurie A

    2012-09-01

    Full Text Available Abstract Background Type II antifreeze protein (AFP from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved. Results Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes. Conclusions These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between ‘higher’ eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

  12. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

    NARCIS (Netherlands)

    van Beusechem, VW; van Rijswijk, ALCT; van Es, HHG; Haisma, HJ; Pinedo, HM; Gerritsen, WR

    2000-01-01

    Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfil

  13. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

    Science.gov (United States)

    Ferrari, S; Kitson, C; Farley, R; Steel, R; Marriott, C; Parkins, D A; Scarpa, M; Wainwright, B; Evans, M J; Colledge, W H; Geddes, D M; Alton, E W

    2001-09-01

    Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

  14. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

    Science.gov (United States)

    Ferrari, S; Kitson, C; Farley, R; Steel, R; Marriott, C; Parkins, D A; Scarpa, M; Wainwright, B; Evans, M J; Colledge, W H; Geddes, D M; Alton, E W

    2001-09-01

    Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal. PMID:11571577

  15. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

    Science.gov (United States)

    Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

    2016-01-01

    Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression. PMID:27386255

  16. Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer.

    Science.gov (United States)

    Pickardt, T; Meixner, M; Schade, V; Schieder, O

    1991-02-01

    Shoot tips and epicotyl-segments of Vicia narbonensis were co-cultivated with Agrobacterium tumefaciens strain C58C1 pGV 3850 HPT, carrying a plasmid coding for hygromycin-phosphotransferase. On callus-induction medium containing 60 mg/l hygromycin for selection, approximately 18% of the explants produced hygromycin-resistant callus. After transfer to regeneration-medium these calluses produced hygromycin-resistant and nopaline-positive somatic embryos which could be regenerated to plantlets. The integration of the T-DNA into the plant genome was confirmed by Southern analysis.

  17. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  18. Ancient horizontal gene transfer from bacteria enhances biosynthetic capabilities of fungi.

    Directory of Open Access Journals (Sweden)

    Imke Schmitt

    Full Text Available BACKGROUND: Polyketides are natural products with a wide range of biological functions and pharmaceutical applications. Discovery and utilization of polyketides can be facilitated by understanding the evolutionary processes that gave rise to the biosynthetic machinery and the natural product potential of extant organisms. Gene duplication and subfunctionalization, as well as horizontal gene transfer are proposed mechanisms in the evolution of biosynthetic gene clusters. To explain the amount of homology in some polyketide synthases in unrelated organisms such as bacteria and fungi, interkingdom horizontal gene transfer has been evoked as the most likely evolutionary scenario. However, the origin of the genes and the direction of the transfer remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We used comparative phylogenetics to infer the ancestor of a group of polyketide synthase genes involved in antibiotic and mycotoxin production. We aligned keto synthase domain sequences of all available fungal 6-methylsalicylic acid (6-MSA-type PKSs and their closest bacterial relatives. To assess the role of symbiotic fungi in the evolution of this gene we generated 24 6-MSA synthase sequence tags from lichen-forming fungi. Our results support an ancient horizontal gene transfer event from an actinobacterial source into ascomycete fungi, followed by gene duplication. CONCLUSIONS/SIGNIFICANCE: Given that actinobacteria are unrivaled producers of biologically active compounds, such as antibiotics, it appears particularly promising to study biosynthetic genes of actinobacterial origin in fungi. The large number of 6-MSA-type PKS sequences found in lichen-forming fungi leads us hypothesize that the evolution of typical lichen compounds, such as orsellinic acid derivatives, was facilitated by the gain of this bacterial polyketide synthase.

  19. Organic farming and gene transfer from genetically modified crops

    OpenAIRE

    Moyes, Catherine L.; Dale, Philip J.

    1999-01-01

    This is the final report of MAFF/Defra project OF0157. Genetically modified (GM) crops cannot be released into the environment and used as food, feed, medicines or industrial processing before they have passed through a rigorous and internationally recognised regulatory process designed to protect human and animal health, and the environment. The UK body that oversees standards in organic farming, the United Kingdom Register of Organic Food Standards (UKROFS), has ruled that gene...

  20. Modulation of lung development by In utero gene transfer

    OpenAIRE

    Santos, Sílvia Gonzaga da Silva

    2009-01-01

    Tese de doutoramento em Ciências da Saúde (ramo de conhecimento em Ciências Biológicas e Biomédicas) Advances in prenatal diagnosis of genetic and congenital disorders with progressively more sensitive techniques may increase opportunities for consideration of prenatal gene therapy. There are a number of genetic and acquired disorders with peri or postnatal pulmonary manifestations. These include monogenetic diseases like cystic fibrosis or surfactant protein B deficiency that wou...

  1. Microbubble-Enhanced Ultrasound Gene Transfer into Fibroblast Cells

    Science.gov (United States)

    Hirayama, Kota; Kaneko, Yukio; Tei, Yuichi; Matsumoto, Yoichiro

    2007-05-01

    Ultrasound finds many applications in the medical field, including ultrasound imaging, non-invasive treatment of tumors and lithotripsy. Ultrasound also has a potential to deliver some therapeutic materials, such as genes, drugs or proteins into cells. It is known that microbubbles can improve the delivery efficiency. It is believed that therapeutic materials can pass through the cell membrane whose permeability is increased by microbubble destruction or the ultrasound pressure. In this study, we investigated the delivery of GFP plasmid gene into the fibroblast cells. Ultrasound (frequency = 2.1 MHz, duty cycle = 10%) was used to irradiate the cultured cells through a medium that contains microbubbles and GFP plasmid. GFP plasmid transfection could be easily observed by fluorescence microscopy. Ultrasound irradiation under a variety of conditions resulted in successful GFP plasmid delivery. Microbubbles enhanced GFP transfection, and conclusions were drawn as to the relationship between gene transfection and various ultrasound exposure parameters. We also investigated the effect of ultrasound intensity on cell viability.

  2. Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure

    DEFF Research Database (Denmark)

    Molin, Søren; Tolker-Nielsen, Tim

    2003-01-01

    There has been much interest in bioremediation based on the introduction of bacteria able to catabolise recalcitrant compounds deposited in the environment. In particular, the delivery of catabolic information in the form of conjugative plasmids to bacterial populations in situ has great potentia...... cycle and released DNA stabilises the biofilm structure. Both of these gene-transfer mechanisms may be autocatalytically promoted in biofilms, presenting new possibilities for efficient bio-enhancement strategies........ As most bacteria in the environment live in surface-associated communities (biofilms), the gene transfer systems within these communities need to be better characterised for bio-enhancement strategies to be developed. Recent findings suggest that gene transfer does take place within biofilms, but studies...

  3. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  4. Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

    OpenAIRE

    Brady, G.; Funk, A.; Mattern, J.; Schütz, G; Brown, R.

    1985-01-01

    Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid....

  5. Potential transfer of extended spectrum β-lactamase encoding gene, blashv18 gene, between Klebsiella pneumoniae in raw foods.

    Science.gov (United States)

    Jung, Yangjin; Matthews, Karl R

    2016-12-01

    This study investigated the transfer frequency of the extended-spectrum β-lactamase-encoding gene (blaSHV18) among Klebsiella pneumoniae in tryptic soy broth (TSB), pasteurized milk, unpasteurized milk, alfalfa sprouts and chopped lettuce at defined temperatures. All transconjugants were characterized phenotypically and genotypically. KP04(ΔKM) and KP08(ΔKM) isolated from seed sprouts and KP342 were used as recipients in mating experiments with K. pneumoniae ATCC 700603 serving as the donor. In mating experiments, no transconjugants were detected at 4 °C in liquid media or chopped lettuce, but detected in all media tested at 15 °C, 24 °C, and 37 °C. At 24 °C, the transfer of blaSHV18 gene occurred more frequently in alfalfa sprouts (5.15E-04 transconjugants per recipient) and chopped lettuce (3.85E-05) than liquid media (1.08E-05). On chopped lettuce, transconjugants were not detected at day 1 post-mating at 15 °C, but observed on day 2 (1.43E-05). Transconjugants carried the blaSHV18 gene transferred from the donor and the virulence gene harbored by recipient. More importantly, a class 1 integrase gene and resistance to tetracycline, trimethoprim/sulfamethoxazole were co-transferred during mating. These quantitative results suggest that fresh produce exposed to temperature abuse may serve as a competent vehicle for the spread of gene encoding for antibiotic resistance, having a potential negative impact on human health. PMID:27554144

  6. Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

    Science.gov (United States)

    Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

    2016-06-01

    Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

  7. Gene transfer for inherited metabolic disorders of the liver: immunological challenges.

    Science.gov (United States)

    Gordts, Stephanie C; Van Craeyveld, Eline; Jacobs, Frank; De Geest, Bart

    2011-01-01

    Hepatocytes are a key target for gene transfer directed at correction of inborn errors of metabolism. The theoretical potential of hepatocyte-directed gene transfer contrasts with the hurdles for clinical translation of this technology. Innate immune responses following gene transfer are initiated by recognition of pathogen-associated molecular patterns by pattern recognition receptors like Toll-like receptors. Adaptive immune responses may constitute the most significant hurdle for efficient gene transfer. Besides the challenge imposed by adaptive immune responses against the vector and the potential problem of pre-existing immunity, immune responses against the transgene product may also constitute an obstacle. The liver is a tolerogenic organ. Naive T cells encounter liver antigens initially in the liver, rather than in lymphoid tissue. Lymph nodes and the spleen are anatomical compartments that provide a particular microarchitecture and microenvironment for the induction of immunity. In contrast, antigen presentation in the liver takes place in a completely different microarchitecture and microenvironment. This is a key aspect of the hepatic adaptive immune tolerance induction. Consistent with the tolerogenic nature of the liver microenvironment, the risk of antibody formation against the transgene product may be limited in the setting of hepatocyte-directed gene transfer and specifically by restricting transgene expression to hepatocytes by use of hepatocyte-specific expression cassettes. However, it is unclear to which extent animal experimental data following gene transfer predict immune responses in humans. Extrapolations from animals to humans are required but should be performed with sufficient insight into the dramatic species differences of the immune system.

  8. Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

    Science.gov (United States)

    Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

    2016-06-01

    Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine. PMID:26683492

  9. RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%~ 72.5%) than in supernatant system (33.1%~ 46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

  10. Transferring Gus gene into intact rice cells by low energy ion beam

    Science.gov (United States)

    Zengliang, Yu; Jianbo, Yang; Yuejin, Wu; Beijiu, Cheng; Jianjun, He; Yuping, Huo

    1993-06-01

    A new technique of transferring genes by low energy ion beam has been reported in this paper. The Gus and CAT (chloramphenicol acetyltransferase) genes, as "foreign" genetic materials, were introduced into the suspension cells and ripe embryos or rice by implantation of 20-30 keV Ar + at doses ranging from 1 × 10 15 to 4 × 10 15 ions/cm 2. The activities of CAT and Gus were detected in the cells and embryos after several weeks. The results indicate that the transfer was a success.

  11. Origin of the plant Tm-1-like gene via two independent horizontal transfer events and one gene fusion event.

    Science.gov (United States)

    Yang, Zefeng; Liu, Li; Fang, Huimin; Li, Pengcheng; Xu, Shuhui; Cao, Wei; Xu, Chenwu; Huang, Jinling; Zhou, Yong

    2016-01-01

    The Tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a direct inhibitor of ToMV RNA replication to protect tomato from infection. The plant Tm-1-like (Tm-1L) protein is predicted to contain an uncharacterized N-terminal UPF0261 domain and a C-terminal TIM-barrel signal transduction (TBST) domain. Homologous searches revealed that proteins containing both of these two domains are mainly present in charophyte green algae and land plants but absent from glaucophytes, red algae and chlorophyte green algae. Although Tm-1 homologs are widely present in bacteria, archaea and fungi, UPF0261- and TBST-domain-containing proteins are generally encoded by different genes in these linages. A co-evolution analysis also suggested a putative interaction between UPF0261- and TBST-domain-containing proteins. Phylogenetic analyses based on homologs of these two domains revealed that plants have acquired UPF0261- and TBST-domain-encoding genes through two independent horizontal gene transfer (HGT) events before the origin of land plants from charophytes. Subsequently, gene fusion occurred between these two horizontally acquired genes and resulted in the origin of the Tm-1L gene in streptophytes. Our results demonstrate a novel evolutionary mechanism through which the recipient organism may acquire genes with functional interaction through two different HGT events and further fuse them into one functional gene. PMID:27647002

  12. Origin of the plant Tm-1-like gene via two independent horizontal transfer events and one gene fusion event.

    Science.gov (United States)

    Yang, Zefeng; Liu, Li; Fang, Huimin; Li, Pengcheng; Xu, Shuhui; Cao, Wei; Xu, Chenwu; Huang, Jinling; Zhou, Yong

    2016-01-01

    The Tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a direct inhibitor of ToMV RNA replication to protect tomato from infection. The plant Tm-1-like (Tm-1L) protein is predicted to contain an uncharacterized N-terminal UPF0261 domain and a C-terminal TIM-barrel signal transduction (TBST) domain. Homologous searches revealed that proteins containing both of these two domains are mainly present in charophyte green algae and land plants but absent from glaucophytes, red algae and chlorophyte green algae. Although Tm-1 homologs are widely present in bacteria, archaea and fungi, UPF0261- and TBST-domain-containing proteins are generally encoded by different genes in these linages. A co-evolution analysis also suggested a putative interaction between UPF0261- and TBST-domain-containing proteins. Phylogenetic analyses based on homologs of these two domains revealed that plants have acquired UPF0261- and TBST-domain-encoding genes through two independent horizontal gene transfer (HGT) events before the origin of land plants from charophytes. Subsequently, gene fusion occurred between these two horizontally acquired genes and resulted in the origin of the Tm-1L gene in streptophytes. Our results demonstrate a novel evolutionary mechanism through which the recipient organism may acquire genes with functional interaction through two different HGT events and further fuse them into one functional gene.

  13. Nano-vectors for efficient liver specific gene transfer

    Directory of Open Access Journals (Sweden)

    Atul Pathak

    2008-03-01

    Full Text Available Atul Pathak1, Suresh P Vyas2, Kailash C Gupta11Nucleic Acids Research Laboratory, Institute of Genomics and Integrative Biology, Delhi  University Campus, Delhi, India 2Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar, (M.P., IndiaAbstract: Recent progress in nanotechnology has triggered the site specific drug/gene delivery research and gained wide acknowledgment in contemporary DNA therapeutics. Amongst various organs, liver plays a crucial role in various body functions and in addition, the site is a primary location of metastatic tumor growth. In past few years, a plethora of nano-vectors have been developed and investigated to target liver associated cells through receptor mediated endocytosis. This emerging paradigm in cellular drug/gene delivery provides promising approach to eradicate genetic as well as acquired diseases affecting the liver. The present review provides a comprehensive overview of potential of various delivery systems, viz., lipoplexes, liposomes, polyplexes, nanoparticles and so forth to selectively relocate foreign therapeutic DNA into liver specific cell type via the receptor mediated endocytosis. Various receptors like asialoglycoprotein receptors (ASGP-R provide unique opportunity to target liver parenchymal cells. The results obtained so far reveal tremendous promise and offer enormous options to develop novel DNAbased pharmaceuticals for liver disorders in near future.Keywords: hepatocytes, nanoparticles, liposomes, nucleic acids, asialoglycoprotein receptors

  14. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

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    Xie Jiatao

    2011-09-01

    Full Text Available Abstract Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses via horizontal gene transfer (HGT. It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

  15. Regulatory and ethical issues for phase I in utero gene transfer studies.

    Science.gov (United States)

    Strong, Carson

    2011-11-01

    Clinical gene transfer research has involved adult and child subjects, and it is expected that gene transfer in fetal subjects will occur in the future. Some genetic diseases have serious adverse effects on the fetus before birth, and there is hope that prenatal gene therapy could prevent such disease progression. Research in animal models of prenatal gene transfer is actively being pursued. The prospect of human phase I in utero gene transfer studies raises important regulatory and ethical issues. One issue not previously addressed arises in applying U.S. research regulations to such studies. Specifically, current regulations state that research involving greater than minimal risk to the fetus and no prospect of direct benefit to the fetus or pregnant woman is not permitted. Phase I studies will involve interventions such as needle insertions through the uterus, which carry risks to the fetus including spontaneous abortion and preterm birth. It is possible that these risks will be regarded as exceeding minimal. Also, some regard the probability of therapeutic benefit in phase I studies to be so low that these studies do not satisfy the regulatory requirement that they "hold out the prospect of direct benefit" to subjects. On the basis of these considerations, investigators and institutional review boards might reasonably conclude that some phase I in utero studies are not to be permitted. This paper identifies considerations that are relevant to such judgments and explores ethically acceptable ways in which phase I studies can be designed so that they are permitted by the regulations. PMID:21846200

  16. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    Directory of Open Access Journals (Sweden)

    Amanda Donnelly

    2015-12-01

    Full Text Available The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i provides enhanced phage-mediated gene transfer; (ii is applicable for laboratory transfection processes and (iii shows promise within industry for large-scale gene transfer applications.

  17. Collective evolution of cyanobacteria and cyanophages mediated by horizontal gene transfer

    Science.gov (United States)

    Shih, Hong-Yan; Rogers, Tim; Goldenfeld, Nigel

    We describe a model for how antagonistic predator-prey coevolution can lead to mutualistic adaptation to an environment, as a result of horizontal gene transfer. Our model is a simple description of ecosystems such as marine cyanobacteria and their predator cyanophages, which carry photosynthesis genes. These genes evolve more rapidly in the virosphere than the bacterial pan-genome, and thus the bacterial population could potentially benefit from phage predation. By modeling both the barrier to predation and horizontal gene transfer, we study this balance between individual sacrifice and collective benefits. The outcome is an emergent mutualistic coevolution of improved photosynthesis capability, benefiting both bacteria and phage. This form of multi-level selection can contribute to niche stratification in the cyanobacteria-phage ecosystem. This work is supported in part by a cooperative agreement with NASA, Grant NNA13AA91A/A0018.

  18. Extensive inter-domain lateral gene transfer in the evolution of the human commensal Methanosphaera stadtmanae

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    Mor Nadia Lurie-Weinberger

    2012-09-01

    Full Text Available Methanosphaera stadtmanae is a commensal methanogenic archaeon found in the human gut. As most of its niche-neighbors are bacteria, it is expected that lateral gene transfer (LGT from bacteria might have contributed to the evolutionary history of this organism. We performed a phylogenomic survey of putative lateral gene transfer events in M. stadtmanae, using a phylogenetic pipeline. Our analysis indicates that a substantial fraction of the proteins of M. stadtmanae are inferred to have been involved in inter-domain LGT. Laterally acquired genes have had a large contribution to surface functions, by providing novel glycosyltransferase functions. In addition, several ABC transporters seem to be of bacterial origin, including the molybdate transporter. Thus, bacterial genes contributed to the adaptation of M. stadtmanae to a host dependent lifestyle by allowing a larger variation in surface structures and increasing transport efficiency in the gut niche which is diverse and competitive

  19. Adenoviral transfer of human interleukin-10 gene in lethal pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Zi-Qian Chen; Yao-Qing Tang; Yi Zhang; Zhi-Hong Jiang; En-Qiang Mao; Wei-Guo Zou; Ruo-Qing Lei; Tian-Quan Han; Sheng-Dao Zhang

    2004-01-01

    AIM: To evaluate the therapeutic effect of adenoviral-vectordelivered human interleukin-10 (hIL-10) gene on severe acute pancreatitis (SAP) rats.METHODS: Healthy Sprague-Dawley (SD) rats were intraperitoneally injected with adenoviral IL-10 gene (AdvhIL-10), empty vector (Adv0) or PBS solution. Blood,liver, pancreas and lung were harvested on the second day to examine hIL-10 level by ELISA and serum amylase by enzymatic assay. A SAP model was induced by retrograde injection of sodium taurocholate through pancreatic duct.SAP rats were then administered with AdvhIL-10, Adv0 and PBS solution by a single intraperitoneal injection 20 min after SAP induction. In addition to serum amylase assay,levels of hIL-10 and tumor necrosis factor-α (TNF-α) were detected by RT-PCR, ELISA and histological study. The mortality rate was studied and analyzed by Kaplan-Meier and log rank analysis.RESULTS: The levels of hIL-10 in the pancreas, liver and lung of healthy rats increased significantly after AdvhIL-10injection (1.42 ng/g in liver, 0.91 ng/g in pancreas); while there was no significant change of hIL-10 in the other two control groups. The concentration of hIL-10 was increased significantly in the SAP rats after AdvhIL-10 injection (1.68 ng/g in liver, 1.12 ng/g in pancreas) compared to the other two SAP groups with blank vector or PBS treatment (P<0.05). The serum amylase levels remained normal in the AdvhIL-10 transfected healthy rats. However,the serum amylase level was significantly elevated in the other two control SAP rats. In contrast, serum amylase was down-regulated in the AdvhIL-10 treated SAP groups.The TNF-α expression in the AdvhIL-10 treated SAP rats was significantly lower compared to the other two control SAP groups. The pathohistological changes in the AdvhIL-10 treated group were better than those in the other two control groups. Furthermore, the mortality of the AdvhIL-10 treated group was significantly reduced compared to the other two control groups (P

  20. Extensive horizontal transfer of core genome genes between two Lactobacillus species found in the gastrointestinal tract

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    Maguin Emmanuelle

    2007-08-01

    Full Text Available Abstract Background While genes that are conserved between related bacterial species are usually thought to have evolved along with the species, phylogenetic trees reconstructed for individual genes may contradict this picture and indicate horizontal gene transfer. Individual trees are often not resolved with high confidence, however, and in that case alternative trees are generally not considered as contradicting the species tree, although not confirming it either. Here we conduct an in-depth analysis of 401 protein phylogenetic trees inferred with varying levels of confidence for three lactobacilli from the acidophilus complex. At present the relationship between these bacteria, isolated from environments as diverse as the gastrointestinal tract (Lactobacillus acidophilus and Lactobacillus johnsonii and yogurt (Lactobacillus delbrueckii ssp. bulgaricus, is ambiguous due to contradictory phenotypical and 16S rRNA based classifications. Results Among the 401 phylogenetic trees, those that could be reconstructed with high confidence support the 16S-rRNA tree or one alternative topology in an astonishing 3:2 ratio, while the third possible topology is practically absent. Lowering the confidence threshold for trees to be taken into consideration does not significantly affect this ratio, and therefore suggests that gene transfer may have affected as much as 40% of the core genome genes. Gene function bias suggests that the 16S rRNA phylogeny of the acidophilus complex, which indicates that L. acidophilus and L. delbrueckii ssp. bulgaricus are the closest related of these three species, is correct. A novel approach of comparison of interspecies protein divergence data employed in this study allowed to determine that gene transfer most likely took place between the lineages of the two species found in the gastrointestinal tract. Conclusion This case-study reports an unprecedented level of phylogenetic incongruence, presumably resulting from extensive

  1. Adaptive horizontal transfer of a bacterial gene to an invasive insect pest of coffee

    OpenAIRE

    Acuña, Ricardo; Padilla, Beatriz E.; Flórez-Ramos, Claudia P.; Rubio, José D.; Herrera, Juan C; Benavides, Pablo; Lee, Sang-Jik; Yeats, Trevor H.; Egan, Ashley N.; Doyle, Jeffrey J.; Rose, Jocelyn K. C.

    2012-01-01

    Horizontal gene transfer (HGT) involves the nonsexual transmission of genetic material across species boundaries. Although often detected in prokaryotes, examples of HGT involving animals are relatively rare, and any evolutionary advantage conferred to the recipient is typically obscure. We identified a gene (HhMAN1) from the coffee berry borer beetle, Hypothenemus hampei, a devastating pest of coffee, which shows clear evidence of HGT from bacteria. HhMAN1 encodes a mannanase, representing a...

  2. Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer.

    Science.gov (United States)

    Ma, Yongjie; Gao, Mingming; Liu, Dexi

    2016-01-01

    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders. PMID:27184920

  3. Gene Transfer and the Reconstruction of Life's Early History from Genomic Data

    Science.gov (United States)

    Gogarten, J. Peter; Fournier, Gregory; Zhaxybayeva, Olga

    2008-03-01

    The metaphor of the unique and strictly bifurcating tree of life, suggested by Charles Darwin, needs to be replaced (or at least amended) to reflect and include processes that lead to the merging of and communication between independent lines of descent. Gene histories include and reflect processes such as gene transfer, symbioses and lineage fusion. No single molecule can serve as a proxy for the tree of life. Individual gene histories can be reconstructed from the growing molecular databases containing sequence and structural information. With some simplifications these gene histories can be represented by furcating trees; however, merging these gene histories into web-like organismal histories, including the transfer of metabolic pathways and cell biological innovations from now-extinct lineages, has yet to be accomplished. Because of these difficulties in interpreting the record retained in molecular sequences, correlations with biochemical fossils and with the geological record need to be interpreted with caution. Advances to detect and pinpoint transfer events promise to untangle at least a few of the intertwined histories of individual genes within organisms and trace them to the organismal ancestors. Furthermore, analysis of the shape of molecular phylogenetic trees may point towards organismal radiations that might reflect early mass extinction events that occurred on a planetary scale.

  4. Detection of horizontal transfer of individual genes by anomalous oligomer frequencies

    Directory of Open Access Journals (Sweden)

    Elhai Jeff

    2012-06-01

    Full Text Available Abstract Background Understanding the history of life requires that we understand the transfer of genetic material across phylogenetic boundaries. Detecting genes that were acquired by means other than vertical descent is a basic step in that process. Detection by discordant phylogenies is computationally expensive and not always definitive. Many have used easily computed compositional features as an alternative procedure. However, different compositional methods produce different predictions, and the effectiveness of any method is not well established. Results The ability of octamer frequency comparisons to detect genes artificially seeded in cyanobacterial genomes was markedly increased by using as a training set those genes that are highly conserved over all bacteria. Using a subset of octamer frequencies in such tests also increased effectiveness, but this depended on the specific target genome and the source of the contaminating genes. The presence of high frequency octamers and the GC content of the contaminating genes were important considerations. A method comprising best practices from these tests was devised, the Core Gene Similarity (CGS method, and it performed better than simple octamer frequency analysis, codon bias, or GC contrasts in detecting seeded genes or naturally occurring transposons. From a comparison of predictions with phylogenetic trees, it appears that the effectiveness of the method is confined to horizontal transfer events that have occurred recently in evolutionary time. Conclusions The CGS method may be an improvement over existing surrogate methods to detect genes of foreign origin.

  5. Horizontal Gene Transfer of the Non-ribosomal Peptide Synthetase Gene Among Endophytic and Epiphytic Bacteria Associated with Ethnomedicinal Plants.

    Science.gov (United States)

    Nongkhlaw, Fenella Mary War; Joshi, S R

    2016-01-01

    This study genetically screened endophytic and epiphytic bacteria associated with ethnomedicinal plants for the presence of the non-ribosomal peptide synthetase (NRPS) gene and identified horizontal gene transfer (HGT) of the NRPS gene between the bacterial species. NRPSs are large multimodular enzymes that synthesize a wide range of biologically active natural compounds that are pharmacologically important. Twenty-nine plant-associated culturable bacteria were screened for the presence of the NRPS gene, of which seven bacterial NRPS gene fragments were successfully detected. According to our findings the presence of NRPS gene among the isolates does not always equate to their antagonistic ability. Phylogenetic analysis of the NRPS and 16S rRNA-encoding genes was used to predict HGT that may have occurred during gene evolution. The occurrence of HGT was demonstrated in the isolates (one inter-phylum and four intra-phyla) and was supported by phylogenetic analysis, mol% G+C content, and tetranucleotide usage pattern and codon usage frequency. Among the four intra-phyla HGT, one isolate showed inter-class HGT and three other isolates showed intra-class HGT.

  6. Horizontal gene transfer and nucleotide compositional anomaly in large DNA viruses

    Directory of Open Access Journals (Sweden)

    Ogata Hiroyuki

    2007-12-01

    Full Text Available Abstract Background DNA viruses have a wide range of genome sizes (5 kb up to 1.2 Mb, compared to 0.16 Mb to 1.5 Mb for obligate parasitic bacteria that do not correlate with their virulence or the taxonomic distribution of their hosts. The reasons for such large variation are unclear. According to the traditional view of viruses as gifted "gene pickpockets", large viral genome sizes could originate from numerous gene acquisitions from their hosts. We investigated this hypothesis by studying 67 large DNA viruses with genome sizes larger than 150 kb, including the recently characterized giant mimivirus. Given that horizontally transferred DNA often have anomalous nucleotide compositions differing from the rest of the genome, we conducted a detailed analysis of the inter- and intra-genome compositional properties of these viruses. We then interpreted their compositional heterogeneity in terms of possible causes, including strand asymmetry, gene function/expression, and horizontal transfer. Results We first show that the global nucleotide composition and nucleotide word usage of viral genomes are species-specific and distinct from those of their hosts. Next, we identified compositionally anomalous (cA genes in viral genomes, using a method based on Bayesian inference. The proportion of cA genes is highly variable across viruses and does not exhibit a significant correlation with genome size. The vast majority of the cA genes were of unknown function, lacking homologs in the databases. For genes with known homologs, we found a substantial enrichment of cA genes in specific functional classes for some of the viruses. No significant association was found between cA genes and compositional strand asymmetry. A possible exogenous origin for a small fraction of the cA genes could be confirmed by phylogenetic reconstruction. Conclusion At odds with the traditional dogma, our results argue against frequent genetic transfers to large DNA viruses from their

  7. Operon Formation is Driven by Co-Regulation and Not by Horizontal Gene Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Price, Morgan N.; Huang, Katherine H.; Arkin, Adam P.; Alm, Eric J.

    2005-04-12

    Although operons are often subject to horizontal gene transfer (HGT), non-HGT genes are particularly likely to be in operons. To resolve this apparent discrepancy and to determine whether HGT is involved in operon formation, we examined the evolutionary history of the genes and operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli (indicating HGTi) form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent with the species tree. In contrast, essential genes and ubiquitous genes without paralogs (genes believed to undergo HGT rarely) often form new operons. We conclude that HGT is not associated with operon formation, but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.

  8. Editing T cell specificity towards leukemia by zinc-finger nucleases and lentiviral gene transfer

    Science.gov (United States)

    Lombardo, Angelo; Magnani, Zulma; Liu, Pei-Qi; Reik, Andreas; Chu, Victoria; Paschon, David E.; Zhang, Lei; Kuball, Jurgen; Camisa, Barbara; Bondanza, Attilio; Casorati, Giulia; Ponzoni, Maurilio; Ciceri, Fabio; Bordignon, Claudio; Greenberg, Philip D.; Holmes, Michael C.; Gregory, Philip D.; Naldini, Luigi; Bonini, Chiara

    2016-01-01

    The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile. PMID:22466705

  9. The impact of gene duplication, insertion, deletion, lateral gene transfer and sequencing error on orthology inference: a simulation study.

    Science.gov (United States)

    Dalquen, Daniel A; Altenhoff, Adrian M; Gonnet, Gaston H; Dessimoz, Christophe

    2013-01-01

    The identification of orthologous genes, a prerequisite for numerous analyses in comparative and functional genomics, is commonly performed computationally from protein sequences. Several previous studies have compared the accuracy of orthology inference methods, but simulated data has not typically been considered in cross-method assessment studies. Yet, while dependent on model assumptions, simulation-based benchmarking offers unique advantages: contrary to empirical data, all aspects of simulated data are known with certainty. Furthermore, the flexibility of simulation makes it possible to investigate performance factors in isolation of one another.Here, we use simulated data to dissect the performance of six methods for orthology inference available as standalone software packages (Inparanoid, OMA, OrthoInspector, OrthoMCL, QuartetS, SPIMAP) as well as two generic approaches (bidirectional best hit and reciprocal smallest distance). We investigate the impact of various evolutionary forces (gene duplication, insertion, deletion, and lateral gene transfer) and technological artefacts (ambiguous sequences) on orthology inference. We show that while gene duplication/loss and insertion/deletion are well handled by most methods (albeit for different trade-offs of precision and recall), lateral gene transfer disrupts all methods. As for ambiguous sequences, which might result from poor sequencing, assembly, or genome annotation, we show that they affect alignment score-based orthology methods more strongly than their distance-based counterparts.

  10. Functional and Evolutionary Characterization of a Gene Transfer Agent’s Multilocus “Genome”

    Science.gov (United States)

    Hynes, Alexander P.; Shakya, Migun; Mercer, Ryan G.; Grüll, Marc P.; Bown, Luke; Davidson, Fraser; Steffen, Ekaterina; Matchem, Heidi; Peach, Mandy E.; Berger, Tim; Grebe, Katherine; Zhaxybayeva, Olga; Lang, Andrew S.

    2016-01-01

    Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell’s genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures “repurposed” by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA “genome” consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA “genome” loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA “genome” is presumably a result of selection on the host organism to retain GTA functionality. PMID:27343288

  11. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    Science.gov (United States)

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  12. Microbubbles and ultrasound increase intraventricular polyplex gene transfer to the brain.

    Science.gov (United States)

    Tan, James-Kevin Y; Pham, Binhan; Zong, Yujin; Perez, Camilo; Maris, Don O; Hemphill, Ashton; Miao, Carol H; Matula, Thomas J; Mourad, Pierre D; Wei, Hua; Sellers, Drew L; Horner, Philip J; Pun, Suzie H

    2016-06-10

    Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits

  13. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  14. Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima

    DEFF Research Database (Denmark)

    Worning, Peder; Jensen, Lars Juhl; Nelson, K. E.;

    2000-01-01

    The recently published complete DNA sequence of the bacterium Thermotoga maritima provides evidence, based on protein sequence conservation, for lateral gene transfer between Archaea and Bacteria. We introduce a new method of periodicity analysis of DNA sequences, based on structural parameters, ...

  15. Current status of gene transfer into haemopoietic progenitor cells: application to Langerhans cell histiocytosis.

    OpenAIRE

    M. Brenner

    1994-01-01

    A number of recent studies have shown that it is possible to obtain significant levels of gene transfer and expression in marrow progenitor cells and their progeny by using retroviral vectors. The data obtained from these studies and the possible applications to Langerhans cell histiocytosis (LCH) are reviewed.

  16. Modifier Genes for Mouse Phosphatidylinositol Transfer Protein alpha (vibrator) That Bypass Juvenile Lethality

    NARCIS (Netherlands)

    Concepcion, Dorothy; Johannes, Frank; Lo, Yuan Hung; Yao, Jay; Fong, Jerry; Hamilton, Bruce A.

    2011-01-01

    Phosphatidylinositol transfer proteins (PITPs) mediate lipid signaling and membrane trafficking in eukaryotic cells. Loss-of-function mutations of the gene encoding PITP alpha in mice result in a range of dosage-sensitive phenotypes, including neurological dysfunction, neurodegeneration, and prematu

  17. The evolution of land plants: a perspective from horizontal gene transfer

    OpenAIRE

    Qia Wang; Hang Sun; Jinling Huang

    2014-01-01

    Recent studies suggest that horizontal gene transfer (HGT) played a significant role in the evolution of eukaryotic lineages. We here review the mechanisms of HGT in plants and the importance of HGT in land plant evolution. In particular, we discuss the role of HGT in plant colonization of land, phototropic response, C4 photosynthesis, and mitochondrial genome evolution.

  18. Assessing the effects of a sequestered germline on interdomain lateral gene transfer in Metazoa.

    Science.gov (United States)

    Jensen, Lindy; Grant, Jessica R; Laughinghouse, Haywood Dail; Katz, Laura A

    2016-06-01

    A sequestered germline in Metazoa has been argued to be an obstacle to lateral gene transfer (LGT), though few studies have specifically assessed this claim. Here, we test the hypothesis that the origin of a sequestered germline reduced LGT events in Bilateria (i.e., triploblast lineages) as compared to early-diverging Metazoa (i.e., Ctenophora, Cnidaria, Porifera, and Placozoa). We analyze single-gene phylogenies generated with over 900 species sampled from among Bacteria, Archaea, and Eukaryota to identify well-supported interdomain LGTs. We focus on ancient interdomain LGT (i.e., those between prokaryotes and multiple lineages of Metazoa) as systematic errors in single-gene tree reconstruction create uncertainties for interpreting eukaryote-to-eukaryote transfer. The breadth of the sampled Metazoa enables us to estimate the timing of LGTs, and to examine the pattern before versus after the evolution of a sequestered germline. We identified 58 LGTs found only in Metazoa and prokaryotes (i.e., bacteria and/or archaea), and seven genes transferred from prokaryotes into Metazoa plus one other eukaryotic clade. Our analyses indicate that more interdomain transfers occurred before the development of a sequestered germline, consistent with the hypothesis that this feature is an obstacle to LGT. PMID:27139503

  19. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam

    2009-01-01

    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  20. Diversity, evolution, and horizontal gene transfer (HGT) in soda lakes

    Science.gov (United States)

    Pinkart, Holly C.; Storrie-Lombardi, Michael C.

    2007-09-01

    Soap Lake is a hypersaline, alkaline lake in Central Washington State (USA). For the past five years the lake has been the site of an NSF Microbial Observatory project devoted to identifying critical geochemical and microbial characteristics of the monimolimnion sediment and water column, and has demonstrated rich multispecies communities occupy all areas of the lake. Soap Lake and similar soda lakes are subject to repeated transient periods of extreme evaporation characterized by significant repetitive alterations in salinity, pH, and total water volume, yet maintain high genetic and metabolic diversity. It has been argued that this repetitive cycle for salinity, alkalinity, and sulfur concentration has been a major driver for prokaryote evolution and diversity. The rapidity of wet-dry cycling places special demands on genome evolution, requirements that are beyond the relatively conservative eukaryotic evolutionary strategy of serial alteration of existing gene sequences in a relatively stable genome. Although HGT is most likely responsible for adding a significant amount of noise to the genetic record, analysis of HGT activity can also provide us with a much-needed probe for exploration of prokaryotic genome evolution and the origin of diversity. Packaging of genetic information within the protective protein capsid of a bacteriophage would seem preferable to exposing naked DNA to the highly alkaline conditions in the lake. In this study, we present preliminary data demonstrating the presence of a diverse group of phage integrases in Soap Lake. Integrase is the viral enzyme responsible for the insertion of phage DNA into the bacterial host's chromosome. The presence of the integrase sequence in bacterial chromosomes is evidence of lysogeny, and the diversity of integrase sequences reported here suggests a wide variety of temperate phage exist in this system, and are especially active in transition zones.

  1. Identification of a Divided Genome for VSH-1, the Prophage-Like Gene Transfer Agent of Brachyspira hyodysenteriae

    Science.gov (United States)

    The Brachyspira hyodysenteriae B204 genome sequence revealed three VSH-1 tail genes hvp31, hvp60, and hvp37, in a 3.6 kb cluster. The location and transcription direction of these genes relative to the previously described VSH-1 16.3 kb gene operon indicate that the gene transfer agent VSH-1 has a ...

  2. Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens:an Assessment of Factors Influencing the Efficiency of Gene Transfer

    Institute of Scientific and Technical Information of China (English)

    Gao Liping; Bao Manzhu

    2004-01-01

    To develop a transformation protocol of Rosa hybrida 'Samantha' via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.5-0.8 for 20 min and co-culture in darkness under 23 °C on medium with 1/2 MS salts and 300 μmol·L-1 AS for 3 d.

  3. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria.

    Science.gov (United States)

    Bennett, P M

    2008-03-01

    Bacteria have existed on Earth for three billion years or so and have become adept at protecting themselves against toxic chemicals. Antibiotics have been in clinical use for a little more than 6 decades. That antibiotic resistance is now a major clinical problem all over the world attests to the success and speed of bacterial adaptation. Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Acquisition of genes needed to elaborate the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids, transposable elements and integron systems, that move genes from one DNA system to another and from one bacterial cell to another, not necessarily one related to the gene donor. Bacterial plasmids serve as the scaffold on which are assembled arrays of antibiotic resistance genes, by transposition (transposable elements and ISCR mediated transposition) and site-specific recombination mechanisms (integron gene cassettes).The evidence suggests that antibiotic resistance genes in human bacterial pathogens originate from a multitude of bacterial sources, indicating that the genomes of all bacteria can be considered as a single global gene pool into which most, if not all, bacteria can dip for genes necessary for survival. In terms of antibiotic resistance, plasmids serve a central role, as the vehicles for resistance gene capture and their subsequent dissemination. These various aspects of bacterial resistance to antibiotics will be explored in this presentation. PMID:18193080

  4. Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications.

    Science.gov (United States)

    Wong, Liang-Fong; Goodhead, Lucy; Prat, Christine; Mitrophanous, Kyriacos A; Kingsman, Susan M; Mazarakis, Nicholas D

    2006-01-01

    The management of disorders of the nervous system remains a medical challenge. The key goals are to understand disease mechanisms, to validate therapeutic targets, and to develop new therapeutic strategies. Viral vector-mediated gene transfer can meet these goals and vectors based on lentiviruses have particularly useful features. Lentiviral vectors can deliver 8 kb of sequence, they mediate gene transfer into any neuronal cell type, expression and therapy are sustained, and normal cellular functions in vitro and in vivo are not compromised. After delivery into the nervous system they induce no significant immune responses, there are no unwanted side effects of the vectors per se to date, and manufacturing and safety testing for clinical applications are well advanced. There are now numerous examples of effective long-term treatment of animal models of neurological disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, motor neuron diseases, lysosomal storage diseases, and spinal injury, using a range of therapeutic genes expressed in lentiviral vectors. Significant issues remain in some areas of neural gene therapy including defining the optimum therapeutic gene(s), increasing the specificity of delivery, regulating expression of potentially toxic genes, and designing clinically relevant strategies. We discuss the applications of lentiviral vectors in therapy and research and highlight the essential features that will ensure their translation to the clinic in the near future. PMID:16409120

  5. Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements.

    Directory of Open Access Journals (Sweden)

    Erik Kristiansson

    Full Text Available The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

  6. The effect of interleukin-6 gene transfer on human cord blood megakaryopoiesis

    Institute of Scientific and Technical Information of China (English)

    Yang Xingsheng; Hitoshi Kurata; Kazuyuki Fujita; Kenichi Tanaka

    2004-01-01

    Objective:To investigate the effect of IL-6 gene transfer into human cord blood hematopoietic stem cells on the production of megakaryocytic progenitors. Methods: IL-6 gene was transfected into human cord blood CD34 + cells using a retrovirus vector with the aid of recombinant fibronectin fragments in the presence of a cocktail of cytokines (SCF, IL-6, sIL-6R, FL, and TPO). Colony-forming units-megakaryocyte (CFU-MK) assays were perfonned as IL-6 gene transduced CD34 + cells were incubated alone or in combination with IL-3 or sIL-6R, controlled with neoR gene transduced CD34 + cells. Results: IL-6 alone or sIL-6R alone stimulated few CFU-MK colonies, the addition of sIL-6R to IL-6 gene transduced CD34 + cells significantly enhanced the production of CFU-MK colonies. IL-6 gene transduced CD34 + cells showed a modest synergistic effect with IL-3. Conclusion: These results suggest that IL-6 gene transfer may protect patients from chemotherapy-induced thrombocytopenia.

  7. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    Science.gov (United States)

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  8. Chromosomal nif Genes Transfer by Conjugation in Nitrogen Fixing Azotobacter chroococcum to Lactobacillus plantarium

    Directory of Open Access Journals (Sweden)

    Adel Kamal Khider

    2011-03-01

    Full Text Available To determine the possibility of transferring chromosomal nitrogen fixation genes (nif genes from Azotobacter chroococcum to Lactobacillus planetarium, a total of 72 Azotobacter chroococcum isolated from Erbil governorate, Iraq were culturally, morphologically and biochemically characterized. Genes for atmospheric nitrogen fixation, located on the chromosome of Azotobacter chroococcum isolates were transferred by conjugation process to a recipient Lactobacillus plantarium isolated from Erbil city soils. The chromosomal genes transferred were verified by analysis of the genomes of donor, recipient and putative transconjugants, by polymorphism of DNA bands obtained through amplification of nifH1, nifH2, nifH3, nifU and nifV genes by PCR technique. The transconjugant cells promote an efficient fixation of nitrogen in liquid cultures fixed 0.2% nitrogen, and in the soil as inoculums of wheat plants, fixed 0.31% nitrogen and solublized 11.71 ppm phosphorus, beside all advantages of Lactic acid bacteria, and probably to be used as inoculums for both nitrogen fixation and solublizing insoluble phosphorus components, and used as biofertilizers

  9. Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

    Directory of Open Access Journals (Sweden)

    Rounak Nande

    Full Text Available Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced of feline fibrosarcomas.

  10. Source-sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities.

    Science.gov (United States)

    Hall, James P J; Wood, A Jamie; Harrison, Ellie; Brockhurst, Michael A

    2016-07-19

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (Hg(R)) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable Hg(R) captured to the chromosome in P. putida A simple mathematical model suggests these differences were likely due to pQBR57's lower intraspecific conjugation rate in P. putida By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source-sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal Hg(R) in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

  11. Source–sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities

    Science.gov (United States)

    Wood, A. Jamie

    2016-01-01

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (HgR) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable HgR captured to the chromosome in P. putida. A simple mathematical model suggests these differences were likely due to pQBR57’s lower intraspecific conjugation rate in P. putida. By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source–sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal HgR in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

  12. Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments

    Directory of Open Access Journals (Sweden)

    Alejandra Moreno-Letelier

    2011-01-01

    Full Text Available The high affinity phosphate transport system (pst is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events.

  13. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

    Directory of Open Access Journals (Sweden)

    Qizhi Liu

    Full Text Available Retrovirus (RV is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.

  14. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  15. SUMO-1 gene transfer improves cardiac function in a large-animal model of heart failure.

    Science.gov (United States)

    Tilemann, Lisa; Lee, Ahyoung; Ishikawa, Kiyotake; Aguero, Jaume; Rapti, Kleopatra; Santos-Gallego, Carlos; Kohlbrenner, Erik; Fish, Kenneth M; Kho, Changwon; Hajjar, Roger J

    2013-11-13

    Recently, the impact of small ubiquitin-related modifier 1 (SUMO-1) on the regulation and preservation of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2a) function was discovered. The amount of myocardial SUMO-1 is decreased in failing hearts, and its knockdown results in severe heart failure (HF) in mice. In a previous study, we showed that SUMO-1 gene transfer substantially improved cardiac function in a murine model of pressure overload-induced HF. Toward clinical translation, we evaluated in this study the effects of SUMO-1 gene transfer in a swine model of ischemic HF. One month after balloon occlusion of the proximal left anterior descending artery followed by reperfusion, the animals were randomized to receive either SUMO-1 at two doses, SERCA2a, or both by adeno-associated vector type 1 (AAV1) gene transfer via antegrade coronary infusion. Control animals received saline infusions. After gene delivery, there was a significant increase in the maximum rate of pressure rise [dP/dt(max)] that was most pronounced in the group that received both SUMO-1 and SERCA2a. The left ventricular ejection fraction (LVEF) improved after high-dose SUMO-1 with or without SERCA2a gene delivery, whereas there was a decline in LVEF in the animals receiving saline. Furthermore, the dilatation of LV volumes was prevented in the treatment groups. SUMO-1 gene transfer therefore improved cardiac function and stabilized LV volumes in a large-animal model of HF. These results support the critical role of SUMO-1 in SERCA2a function and underline the therapeutic potential of SUMO-1 for HF patients.

  16. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  17. What's next for gene therapy? Virginia Tech plastics researchers design polymer macromolecules as gene transfer agents

    OpenAIRE

    Trulove, Susan

    2006-01-01

    Gene therapy depends upon foreign DNA, even viruses, to deliver genes, therapeutic proteins, or medicine to cells within the body. Many scientists are looking for better chaperones across the cell membrane. Virginia Tech researchers think polymer molecules can be created to do the job.

  18. Lateral Transfer of the Denitrification Pathway Genes among Thermus thermophilus Strains▿

    Science.gov (United States)

    Alvarez, Laura; Bricio, Carlos; José Gómez, Manuel; Berenguer, José

    2011-01-01

    Nitrate respiration is a common and strain-specific property in Thermus thermophilus encoded by the nitrate respiration conjugative element (NCE) that can be laterally transferred by conjugation. In contrast, nitrite respiration and further denitrification steps are restricted to a few isolates of this species. These later steps of the denitrification pathway are under the regulatory control of an NCE-encoded transcription factor, but nothing is known about their coding sequences or its putative genetic linkage to the NCE. In this study we examine the genetic linkage between nitrate and nitrite respiration through lateral gene transfer (LGT) assays and describe a cluster of genes encoding the nitrite-nitric oxide respiration in T. thermophilus PRQ25. We show that the whole denitrification pathway can be transferred from the denitrificant strain PRQ25 to an aerobic strain, HB27, and that the genes coding for nitrite and nitric oxide respiration are encoded near the NCE. Sequence data from the draft genome of PRQ25 confirmed these results and allowed us to describe the most compact nor-nir cluster known thus far and to demonstrate the expression and activities of the encoded enzymes in the HB27 denitrificant derivatives obtained by LGT. We conclude that this NCE nor-nir supercluster constitutes a whole denitrification island that can be spread by lateral transfer among Thermus thermophilus strains. PMID:21169443

  19. Growth factor enhanced retroviral gene transfer to the adult central nervous system.

    Science.gov (United States)

    King, L A; Mitrophanous, K A; Clark, L A; Kim, V N; Rohll, J B; Kingsman, A J; Colello, R J

    2000-07-01

    The use of viral vectors for gene delivery into mammalian cells provides a new approach in the treatment of many human diseases. The first viral vector approved for human clinical trials was murine leukemia virus (MLV), which remains the most commonly used vector in clinical trials to date. However, the application of MLV vectors is limited since MLV requires cells to be actively dividing in order for transduction and therefore gene delivery to occur. This limitation precludes the use of MLV for delivering genes to the adult CNS, where very little cell division is occurring. However, we speculated that this inherent limitation of ML V may be overcome by utilizing the known mitogenic effect of growth factors on cells of the CNS. Specifically, an in vivo application of growth factor to the adult brain, if able to induce cell division, could enhance MLV-based gene transfer to the adult brain. We now show that an exogenous application of basic fibroblast growth factor induces cell division in vivo. Under these conditions, where cells of the adult brain are stimulated to divide, MLV-based gene transfer is significantly enhanced. This novel approach precludes any vector modifications and provides a simple and effective way of delivering genes to cells of the adult brain utilizing MLV-based retroviral vectors. PMID:10918476

  20. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies

  1. Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

    2014-10-03

    Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

  2. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  3. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

    Directory of Open Access Journals (Sweden)

    Julius W Kim

    Full Text Available BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  4. Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.

    Science.gov (United States)

    Boothby, Thomas C; Tenlen, Jennifer R; Smith, Frank W; Wang, Jeremy R; Patanella, Kiera A; Nishimura, Erin Osborne; Tintori, Sophia C; Li, Qing; Jones, Corbin D; Yandell, Mark; Messina, David N; Glasscock, Jarret; Goldstein, Bob

    2015-12-29

    Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

  5. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  6. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    International Nuclear Information System (INIS)

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references

  7. Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Saliou Fall

    Full Text Available Horizontal gene transfer (HGT is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3% of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment

  8. Horizontal gene transfers and cell fusions in microbiology, immunology and oncology (Review).

    Science.gov (United States)

    Sinkovics, Joseph G

    2009-09-01

    Evolving young genomes of archaea, prokaryota and unicellular eukaryota were wide open for the acceptance of alien genomic sequences, which they often preserved and vertically transferred to their descendants throughout three billion years of evolution. Established complex large genomes, although seeded with ancestral retroelements, have come to regulate strictly their integrity. However, intruding retroelements, especially the descendents of Ty3/Gypsy, the chromoviruses, continue to find their ways into even the most established genomes. The simian and hominoid-Homo genomes preserved and accommodated a large number of endogenous retroviral genomic segments. These retroelements may mature into exogenous retroviruses, or into functional new genes. Phages and viruses have been instrumental in incorporating and transferring host cell genes. These events profoundly influenced and altered the course of evolution. Horizontal (lateral) gene transfers (HGT) overwhelmed the genomes of the ancient protocells and the evolving unicellular microorganisms, actually leading to their Cambrian explosion. While the rigidly organized genomes of multicellular organisms increasingly resist H/LGT, de-differentiated cells assuming the metabolism of their onto- or phylogenetic ancestors, open up widely to the practice of H/LGT by direct transfer, or to transfers mediated by viruses, or by cell fusions. This activity is intensified in malignantly transformed cells, thus rendering these subjects receptive to therapy with oncolytic viruses and with viral vectors of tumor-suppressive or immunogenic genetic materials. Naturally formed hybrids of dendritic and tumor cells are often tolerogenic, whereas laboratory products of these unisons may be immunogenic in the hosts of origin. As human breast cancer stem cells are induced by a treacherous class of CD8+ T cells to undergo epithelial to mesenchymal (ETM) transition and to yield to malignant transformation by the omnipresent proto

  9. Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer ability

    Directory of Open Access Journals (Sweden)

    Satoko eNonaka

    2014-12-01

    Full Text Available Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.

  10. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

    Directory of Open Access Journals (Sweden)

    Kaul Rajinder

    2009-11-01

    Full Text Available Abstract Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia

  11. Sequence diversities of serine-aspartate repeat genes among Staphylococcus aureus isolates from different hosts presumably by horizontal gene transfer.

    Directory of Open Access Journals (Sweden)

    Huping Xue

    Full Text Available BACKGROUND: Horizontal gene transfer (HGT is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr family has been compared among different sources of Staphylococcus aureus (S. aureus to discover sequence diversities within their genomes. METHODOLOGY/PRINCIPAL FINDINGS: Four sdr genes were analyzed for 21 different S. aureus strains and 218 mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122 and mastitis isolates in this study, ovine mastitis (ED133, pig (ST398, chicken (ED98, and human methicillin-resistant S. aureus (MRSA (TCH130, MRSA252, Mu3, Mu50, N315, 04-02981, JH1 and JH9 were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. CONCLUSIONS: Certain sdr gene sequences were shared in S. aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may

  12. Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein

    International Nuclear Information System (INIS)

    A high-titer amphotropic retroviral vector containing the neomycin resistance gene and a hybrid γ-β genomic human globin gene has been constructed. Mouse erythroleukemia cells infected with this virus were found to contain the full transcriptional unit of the transferred human globin gene by Southern blot analysis. These cells contain normally initiated, spliced, and terminated human globin mRNA. The human globin mRNA level increased 5- to 10-fold upon induction of the mouse erythroleukemia cells. Human globin chains were produced but only in a fraction of the cells as detected by immunofluorescent staining. A similar retrovirus containing a human β-globin gene was used to transduce mouse erythroleukemia cells resulting in much higher levels of human globin synthesis than detected in mouse erythroleukemia cells transduced with the γ-β globin virus

  13. An adeno-associated virus vector-mediated multiple gene transfer for dopamine synthetic enzymes

    Institute of Scientific and Technical Information of China (English)

    Fan Dongsheng (樊东升); Shen Yang(沈扬)

    2000-01-01

    Objective: To explore a multiple gene transfer approach with separate adeno-associated virus vectors. Methods: The genes of dopamine synthetic enzymes, tyrosine hydroxylasc (TH), GTP cyclohydrolase I (GCH, an enzyme critical for tetrahydrobioptcrin synthesis), and aromatic L-amino acid decarboxylase (AADC), were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, GCH, and AADC were detected by Western blot analysis. L-dopa and dopamine levels in the ceils were assayed by HPLC. Results: TH, GCH, and AADC proteins were effectively cocxpressed in the transduced cells with three separate AAV vectors, AAV-TH, AAV-GCH, and AAV-AADC. Furthermore, the coexpression of these three proteins resulted in an effectively spontaneous dopainc production in the cotransduced cells. Conclusion: The triple transduction of TH, GCH, and AADC genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.

  14. Enhanced horizontal transfer of antibiotic resistance genes in freshwater microcosms induced by an ionic liquid.

    Directory of Open Access Journals (Sweden)

    Qing Wang

    Full Text Available The spread and propagation of antibiotic resistance genes (ARGs is a worldwide public health concern. Ionic liquids (ILs, considered as "environmentally friendly" replacements for industrial organic solvents, have been widely applied in modern industry. However, few data have been collected regarding the potential ecological and environmental risks of ILs, which are important for preparing for their potential discharge into the environment. In this paper, the IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6] (0.001-5.0 g/L was tested for its effects on facilitating ARGs horizontal transfer mediated by plasmid RP4 in freshwater microcosms. In the horizontal transfer microcosms, the transfer frequency of plasmid RP4 was significantly enhanced (60-fold higher than untreated groups by the IL [BMIm][PF6] (1.0 g/L. Meanwhile, two strains of opportunistic pathogen Acinetobacter spp. and Salmonella spp. were isolated among the transconjugants, illustrating plasmid RP4 mediated horizontal transfer of ARGs occurred in pathogen. This could increase the risk of ARGs dissemination to human pathogens and pose great threat to public health. The cause that [BMIm[PF6] enhanced the transfer frequency of plasmid RP4 was proposed by suppressed cell membrane barrier and enhanced cell membrane permeability, which was evidenced by flow cytometry (FCM. This is the first report that some ILs facilitate horizontal transfer of plasmid RP4 which is widely distributed in the environment and thus add the adverse effects of the environmental risk of ILs.

  15. Functional biogeography as evidence of gene transfer in hypersaline microbial communities.

    Directory of Open Access Journals (Sweden)

    J Jacob Parnell

    Full Text Available BACKGROUND: Horizontal gene transfer (HGT plays a major role in speciation and evolution of bacteria and archaea by controlling gene distribution within an environment. However, information that links HGT to a natural community using relevant population-genetics parameters and spatial considerations is scarce. The Great Salt Lake (Utah, USA provides an excellent model for studying HGT in the context of biogeography because it is a contiguous system with dispersal limitations due to a strong selective salinity gradient. We hypothesize that in spite of the barrier to phylogenetic dispersal, functional characteristics--in the form of HGT--expand beyond phylogenetic limitations due to selective pressure. METHODOLOGY AND RESULTS: To assay the functional genes and microorganisms throughout the GSL, we used a 16S rRNA oligonucleotide microarray (Phylochip and a functional gene array (GeoChip to measure biogeographic patterns of nine microbial communities. We found a significant difference in biogeography based on microarray analyses when comparing Sørensen similarity values for presence/absence of function and phylogeny (Student's t-test; p = 0.005. CONCLUSION AND SIGNIFICANCE: Biogeographic patterns exhibit behavior associated with horizontal gene transfer in that informational genes (16S rRNA have a lower similarity than functional genes, and functional similarity is positively correlated with lake-wide selective pressure. Specifically, high concentrations of chromium throughout GSL correspond to an average similarity of chromium resistance genes that is 22% higher than taxonomic similarity. This suggests active HGT may be measured at the population level in microbial communities and these biogeographic patterns may serve as a model to study bacteria adaptation and speciation.

  16. Transfer

    DEFF Research Database (Denmark)

    Wahlgren, Bjarne; Aarkrog, Vibe

    Bogen er den første samlede indføring i transfer på dansk. Transfer kan anvendes som praksis-filosofikum. Den giver en systematisk indsigt til den studerende, der spørger: Hvordan kan teoretisk viden bruges til at reflektere over handlinger i situationer, der passer til min fremtidige arbejdsplads?...

  17. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells.

    Science.gov (United States)

    Hyland, Kendra A; Olson, Erik R; McIvor, R Scott

    2015-10-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34(+) HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon-chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications. PMID:26176276

  18. Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations o...

  19. The Extent and Regulation of Lateral Gene Transfer in Natural Microbial Ecosystems

    DEFF Research Database (Denmark)

    Aminov, Rustam I.

    2012-01-01

    The importance of horizontal gene transfer (HGT) in bacterial evolution is evident from the retrospective analyses of bacterial genomes, which suggest that a substantial part of bacterial genomes is of foreign origin. Another line of evidence that supports the possibility of rapid adaptation...... of bacteria through lateral gene exchange is the history of antibiotic use by humans. Within a very brief period of the 'antibiotic era' many bacterial pathogens were able to acquire the mechanisms allowing them to withstand the selective pressure of antibiotics. And, finally, field and microcosm studies...

  20. Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

    Science.gov (United States)

    Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

    1997-03-01

    Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

  1. Emergence of collective territorial defense in bacterial communities: horizontal gene transfer can stabilize microbiomes.

    Science.gov (United States)

    Juhász, János; Kertész-Farkas, Attila; Szabó, Dóra; Pongor, Sándor

    2014-01-01

    Multispecies bacterial communities such as the microbiota of the gastrointestinal tract can be remarkably stable and resilient even though they consist of cells and species that compete for resources and also produce a large number of antimicrobial agents. Computational modeling suggests that horizontal transfer of resistance genes may greatly contribute to the formation of stable and diverse communities capable of protecting themselves with a battery of antimicrobial agents while preserving a varied metabolic repertoire of the constituent species. In other words horizontal transfer of resistance genes makes a community compatible in terms of exoproducts and capable to maintain a varied and mature metagenome. The same property may allow microbiota to protect a host organism, or if used as a microbial therapy, to purge pathogens and restore a protective environment. PMID:24755769

  2. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

    Science.gov (United States)

    Garbian, Yael; Maori, Eyal; Kalev, Haim; Shafir, Sharoni; Sela, Ilan

    2012-12-01

    The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

  3. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

    Directory of Open Access Journals (Sweden)

    Yael Garbian

    2012-12-01

    Full Text Available The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

  4. Inhibitory effect of Ca2+ on in vivo gene transfer by electroporation

    Institute of Scientific and Technical Information of China (English)

    Yong-gang ZHAO; Hui-li LU; Jin-liang PENG; Yu-hong XU

    2006-01-01

    Aim:To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice.Methods:Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid,with or Without Ca2+ and various other ions.Resuits:For in vivo electroporation,the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression,to less than 5% of control values.Only Ca2+,not other ions,caused inhibition,and the effect was not tissue specific.More surprisingly.even when Ca2+ ions were delivered by electroporation before or after DNA administration,similar effects were still observed.Conelusion:The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific,ie,the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.

  5. Lysophosphatidylcholine as an adjuvant for lentiviral vector mediated gene transfer to airway epithelium: effect of acyl chain length

    Directory of Open Access Journals (Sweden)

    Anson Don S

    2010-06-01

    Full Text Available Abstract Background Poor gene transfer efficiency has been a major problem in developing an effective gene therapy for cystic fibrosis (CF airway disease. Lysophosphatidylcholine (LPC, a natural airway surfactant, can enhance viral gene transfer in animal models. We examined the electrophysiological and physical effect of airway pre-treatment with variants of LPC on lentiviral (LV vector gene transfer efficiency in murine nasal airways in vivo. Methods Gene transfer was assessed after 1 week following nasal instillations of a VSV-G pseudotype LV vector pre-treated with a low and high dose of LPC variants. The electrophysiological effects of a range of LPC variants were assessed by nasal transepithelial potential difference measurements (TPD to determine tight junction permeability. Any physical changes to the epithelium from administration of the LPC variants were noted by histological methods in airway tissue harvested after 1 hour. Results Gene transduction was significantly greater compared to control (PBS for our standard LPC (palmitoyl/stearoyl mixture treatment and for the majority of the other LPC variants with longer acyl chain lengths. The LPC variant heptadecanoyl also produced significantly greater LV gene transfer compared to our standard LPC mixture. LV gene transfer and the transepithelial depolarization produced by the 0.1% LPC variants at 1 hour were strongly correlated (r2 = 0.94, but at the 1% concentration the correlation was less strong (r2 = 0.59. LPC variants that displayed minor to moderate levels of disruption to the airway epithelium were clearly associated with higher LV gene transfer. Conclusions These findings show the LPC variants effect on airway barrier function and their correlation to the effectiveness of gene expression. The enhanced expression produced by a number of LPC variants should provide new options for preclinical development of efficient airway gene transfer techniques.

  6. Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes

    Science.gov (United States)

    Shousha, Amira; Awaiwanont, Nattakarn; Sofka, Dmitrij; Smulders, Frans J. M.; Paulsen, Peter; Szostak, Michael P.; Humphrey, Tom

    2015-01-01

    Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health. PMID:25934615

  7. Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes.

    Science.gov (United States)

    Shousha, Amira; Awaiwanont, Nattakarn; Sofka, Dmitrij; Smulders, Frans J M; Paulsen, Peter; Szostak, Michael P; Humphrey, Tom; Hilbert, Friederike

    2015-07-01

    Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

  8. Transfer of alien genes by means of induced translocation in oats and other crop species

    International Nuclear Information System (INIS)

    Some of the best sources of resistance to mildew, which is the most important disease of the oat crop in the United Kingdom, occur in related weed species. The mildew resistance found in a genotype of the tetraploid species Avena barbata has been transferred into the germ plasm of the cultivated hexaploid species A. sativa by means of an induced translocation. The procedures adopted to isolate the desirable translocation and to determine its breeding behaviour are described. A number of alien genes have been transferred into wheat by means of induced translocations and genetic induction, but their successful introduction into commercial varieties has been limited. In this paper, the use and limitations of alien transfers as breeding material are discussed. (author)

  9. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  10. Emergence of Collective Territorial Defense in Bacterial Communities: Horizontal Gene Transfer Can Stabilize Microbiomes

    OpenAIRE

    János Juhász; Attila Kertész-Farkas; Dóra Szabó; Sándor Pongor

    2014-01-01

    Multispecies bacterial communities such as the microbiota of the gastrointestinal tract can be remarkably stable and resilient even though they consist of cells and species that compete for resources and also produce a large number of antimicrobial agents. Computational modeling suggests that horizontal transfer of resistance genes may greatly contribute to the formation of stable and diverse communities capable of protecting themselves with a battery of antimicrobial agents while preserving ...

  11. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  12. Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation

    OpenAIRE

    Kouzel, Nadzeya; Oldewurtel, Enno R.; Maier, Berenike

    2015-01-01

    Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal t...

  13. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    OpenAIRE

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-pro...

  14. Bayesian analysis of congruence of core genes in Prochlorococcus and Synechococcus and implications on horizontal gene transfer.

    Directory of Open Access Journals (Sweden)

    Nicholas J Matzke

    Full Text Available It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. "Universal proteins" have been used to infer the organismal phylogeny, but have been criticized as being only the "tree of one percent." Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees - a "treecloud" - for each, as well as for a concatenated dataset based on putative "universal proteins." We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS. We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the "tree of one percent" is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.

  15. Effects of laser parameters on propagation characteristics of laser-induced stress wave for gene transfer

    Science.gov (United States)

    Ando, Takahiro; Sato, Shunichi; Terakawa, Mitsuhiro; Ashida, Hiroshi; Obara, Minoru

    2010-02-01

    Laser-based gene delivery is attractive as a new method for topical gene therapy because of the high spatial controllability of laser energy. Previously, we demonstrated that an exogenous gene can be transferred to cells both in vitro and in vivo by applying nanosecond pulsed laser-induced stress waves (LISWs) or photomechanical waves (PMWs). In this study, we investigated effects of laser parameters on the propagation characteristics of LISWs in soft tissue phantoms and depth-dependent properties of gene transfection. Temporal pressure profiles of LISWs were measured with a hydrophone, showing that with a larger laser spot diameter, LISWs can be propagated more efficiently in phantoms with keeping flat wavefront. Phantoms with various thicknesses were placed on the rat dorsal skin that had been injected with plasmid DNA coding for reporter gene, and LISWs were applied from the top of the phantom. Efficient gene expression was observed in the rat skin that had interacted with LISWs propagating through a 15-mm-thick phantom. These results would be useful to determine appropriate laser parameters for gene delivery to deep-located tissue by transcutaneous application of LISWs.

  16. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-05-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  17. Characterization and transfer studies of macrolide resistance genes in Streptococcus pneumoniae from Denmark

    DEFF Research Database (Denmark)

    Nielsen, Karen L; Hammerum, Anette M; Lambertsen, Lotte M;

    2010-01-01

    susceptibility was tested, and they were genotyped and serotyped. Gene transfer was studied for selected isolates. The frequency of erm(B) was significantly higher in non-invasive isolates compared to invasive isolates (p = 0.001). For the first time, mef(I) was detected in 1 isolate in Denmark. All tested mef......(E) isolates had an identical mef(E) sequence, apart from 1 gene with a point mutation, and mef(E) was correlated to 7 different sero-types. The tested erm(B) sequences were 99.3% similar with 5 point mutations at different positions distributed among different serotypes, which did not cause a detectable...... influence on the protein. Transformation was detectable in 5 out of 13 isolates and transfer of erm(B), mef(I) and mef(E) was detected. To our knowledge, this is the first time mef(I) has been proved transformable. Gene transfer by conjugation was not detectable. Erythromycin resistance in pneumococcal...

  18. Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to simulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies

  19. THE RISK OF GENE TRANSFERRING IN THE INSURANCE PROTECTION OF AGRICULTERE

    Directory of Open Access Journals (Sweden)

    M. Malik

    2016-03-01

    Full Text Available The paper justified essence of genetic engineering as the object of insurance services. Defines the concept of risk gene transferring. The character features of this specific risk. The influence and consequences for agricultural producers. The description of the possible creation of the concept of insurance services that cover risk of gene transferring. The study reveals of the use of GMOs in agriculture, due to issues of economic security of a particular region or country as a whole. To determined the impact of risks and control for developing and developed countries that are important aspects of farming. Changes in weather, climate, productivity, price values, public policy, the situation on global markets can cause large fluctuations in agricultural production, and consequently affecting the income of agricultural producers. Risk management includes a range of strategies that reduce the social and financial implications of possible changes affecting the production and income of farmers. There is a need for an in-depth study of the theoretical and practical aspects of the impact of the risk of gene transferring in the context of insurance protection.

  20. Investigating rate-limiting barriers to nanoscale nonviral gene transfer with nanobiophotonics

    Science.gov (United States)

    Chen, Hunter H.

    Nucleic acids are a novel class of therapeutics poised to address many unmet clinical needs. Safe and efficient delivery remains a significant challenge that has delayed the realization of the full therapeutic potential of nucleic acids. Nanoscale nonviral vectors offer an attractive alternative to viral vectors as natural and synthetic polymers or polypeptides may be rationally designed to meet the unique demands of individual applications. A mechanistic understanding of cellular barriers is necessary to develop guidelines for designing custom gene carriers which are expected to greatly impact this delivery challenge. The work herein focused on the relationships among nanocomplex stability, intracellular trafficking and unpacking kinetics, and DNA degradation. Ultrasensitive nanosensors based on QD-FRET were developed to characterize the biophysical properties of nanocomplexes and study these rate-limiting steps. Quantitative image analysis enabled the distributions of the subpopulation of condensed or released DNA to be determined within the major cellular compartments encountered during gene transfer. The steady state stability and unpacking kinetics within these compartments were found to impact transgene expression, elucidating multiple design strategies to achieve efficient gene transfer. To address enzymatic barriers, a novel two-step QD-FRET nanosensor was developed to analyze unpacking and DNA degradation simultaneously, which has not been accomplished previously. Bioresponsive strategies such as disulfide crosslinking and thermosensitivity were evaluated by QD-FRET and quantitative compartmental analysis as case studies to determine appropriate design specifications for thiolated polymers and thermoresponsive polypeptides. Relevant nanobiophotonic tools were developed as a platform to study major rate-limiting barriers to nanomedicine and demonstrated the feasibility of using mechanistic information gained from these tools to guide the rational design of

  1. Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.

  2. 重组人p53腺病毒药物对人喉癌细胞的抑制实验%ADENOVIRUS-MEDIATED P53 GENE THERAPY OF HUMAN LARYNGEAL CANCER

    Institute of Scientific and Technical Information of China (English)

    敖敏; 何刚; 梁传余

    2007-01-01

    [目的]探索p53基因在喉癌基因治疗方面的可行性.[方法]以人喉癌细胞系Hep-2为实验对象,将重组人p53腺病毒药物(rAd/p53)转染Hep-2细胞,体外实验观察重组人p53腺病毒药物(rAd/p53)对Hep-2细胞生长的影响.[结果]各浓度重组人p53 腺病毒药物(rAd/p53)(1010、109、108、107)对Hep-2生长均有抑制.尤以1010明显.转染3d后,重组人p53腺病毒药物(rAd/p53)诱导Hep-2细胞明显凋亡.[结论]重组人p53腺病毒药物(rAd/p53)对Hep-2细胞生长能有效抑制,能明显诱导其凋亡,为喉癌的治疗提供了临床前依据.

  3. Adenovirus-mediated p53 gene therapy in human nasopharyngeal cancer%重组人p53腺病毒基因药物对人鼻咽癌细胞的抑制实验

    Institute of Scientific and Technical Information of China (English)

    敖敏; 何刚

    2010-01-01

    目的 探索p53基因在鼻咽癌基因治疗方面的可行性.方法 以人鼻咽癌CNE细胞株为实验对象,将重组人p53腺病毒药物(1010rAd/p53)转染人鼻咽癌CNE细胞,用MTT比色实验及流式细胞仪实验的方法进行体外实验,观察重组人p53腺病毒药物(rAd/p53)对人鼻咽癌CNE细胞体外生长的影响.结果 各浓度重组人p53腺病毒药物(1010rAd/p53、109rAd/p53、108rAd/p53、107rAd/p53)对人鼻咽癌CNE细胞生长有抑制.尤以1010rAd/p53明显.转染3天后,重组人p53腺病毒药物(rAd/p53)诱导人鼻咽癌CNE细胞明显凋亡.结论 重组人p53腺病毒药物(rAd/p53)对人鼻咽癌CNE细胞生长能有效抑制,为鼻咽癌的基因治疗提供了实验依据.

  4. Airway gene transfer in a non-human primate: lentiviral gene expression in marmoset lungs.

    Science.gov (United States)

    Farrow, N; Miller, D; Cmielewski, P; Donnelley, M; Bright, R; Parsons, D W

    2013-01-01

    Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials. PMID:23412644

  5. Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo

    Institute of Scientific and Technical Information of China (English)

    陈卫昌; 傅建新; 刘强; 阮长耿; 萧树东

    2003-01-01

    Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line, LoVo.Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2×105 CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.Results After transfection and superinfection, amphotropic retrovirus was collected, and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited endothelial cell growth up to 67% (P<0.001), compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group, accompanied by a reduction in vessels, compared with the control group (P<0.01). Conclusion Retroviruses can allow functional expression of the endostatin gene in human colon tumors, showing promise for an antitumor strategy using antiangiogenesis.

  6. Insights on the Horizontal Gene Transfer of Carbapenemase Determinants in the Opportunistic Pathogen Acinetobacter baumannii

    Science.gov (United States)

    Da Silva, Gabriela Jorge; Domingues, Sara

    2016-01-01

    Horizontal gene transfer (HGT) is a driving force to the evolution of bacteria. The fast emergence of antimicrobial resistance reflects the ability of genetic adaptation of pathogens. Acinetobacter baumannii has emerged in the last few decades as an important opportunistic nosocomial pathogen, in part due to its high capacity of acquiring resistance to diverse antibiotic families, including to the so-called last line drugs such as carbapenems. The rampant selective pressure and genetic exchange of resistance genes hinder the effective treatment of resistant infections. A. baumannii uses all the resistance mechanisms to survive against carbapenems but production of carbapenemases are the major mechanism, which may act in synergy with others. A. baumannii appears to use all the mechanisms of gene dissemination. Beyond conjugation, the mostly reported recent studies point to natural transformation, transduction and outer membrane vesicles-mediated transfer as mechanisms that may play a role in carbapenemase determinants spread. Understanding the genetic mobilization of carbapenemase genes is paramount in preventing their dissemination. Here we review the carbapenemases found in A. baumannii and present an overview of the current knowledge of contributions of the various HGT mechanisms to the molecular epidemiology of carbapenem resistance in this relevant opportunistic pathogen.

  7. Horizontal gene transfer events reshape the global landscape of arm race between viruses and homo sapiens.

    Science.gov (United States)

    Chen, Dong-Sheng; Wu, Yi-Quan; Zhang, Wei; Jiang, San-Jie; Chen, Shan-Ze

    2016-01-01

    Horizontal gene transfer (HGT) drives the evolution of recipient organism particularly if it provides a novel function which enhances the fitness or its adaption to the environment. Virus-host co-evolution is attractive for studying co-evolutionary processes, since viruses strictly replicate inside of the host cells and thus their evolution is inexorably tangled with host biology. HGT, as a mechanism of co-evolution between human and viruses, has been widely documented, however, the roles HGT play during the interaction between human and viruses are still in their infancy. In this study, we performed a comprehensive analysis on the genes horizontally transferred between viruses and their corresponding human hosts. Our study suggests that the HGT genes in human are predominantly enriched in immune related GO terms while viral HGT genes are tend to be encoded by viruses which promote the invasion of immune system of hosts. Based on our results, it gives us a hint about the evolution trajectory of HGT events. Overall, our study suggests that the HGT between human and viruses are highly relevant to immune interaction and probably reshaped the arm race between hosts and viruses. PMID:27270140

  8. DNA bar coding and pyrosequencing to analyze adverse events in therapeutic gene transfer.

    Science.gov (United States)

    Wang, Gary P; Garrigue, Alexandrine; Ciuffi, Angela; Ronen, Keshet; Leipzig, Jeremy; Berry, Charles; Lagresle-Peyrou, Chantal; Benjelloun, Fatine; Hacein-Bey-Abina, Salima; Fischer, Alain; Cavazzana-Calvo, Marina; Bushman, Frederic D

    2008-05-01

    Gene transfer has been used to correct inherited immunodeficiencies, but in several patients integration of therapeutic retroviral vectors activated proto-oncogenes and caused leukemia. Here, we describe improved methods for characterizing integration site populations from gene transfer studies using DNA bar coding and pyrosequencing. We characterized 160,232 integration site sequences in 28 tissue samples from eight mice, where Rag1 or Artemis deficiencies were corrected by introducing the missing gene with gamma-retroviral or lentiviral vectors. The integration sites were characterized for their genomic distributions, including proximity to proto-oncogenes. Several mice harbored abnormal lymphoproliferations following therapy--in these cases, comparison of the location and frequency of isolation of integration sites across multiple tissues helped clarify the contribution of specific proviruses to the adverse events. We also took advantage of the large number of pyrosequencing reads to show that recovery of integration sites can be highly biased by the use of restriction enzyme cleavage of genomic DNA, which is a limitation in all widely used methods, but describe improved approaches that take advantage of the power of pyrosequencing to overcome this problem. The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution.

  9. Changes in glucose metabolism and gene expression after transfer of anti-angiogenic genes in rat hepatoma

    International Nuclear Information System (INIS)

    Human troponin I (TROP), the soluble receptor for vascular endothelial growth factor (sFLT) and angiostatin (ASTAT) are potent inhibitors of endothelial cell proliferation, angiogenesis and tumour growth in vivo. Transfer of these genes into tumours may induce changes not only in perfusion, but also more general ones such as changes in metabolism. The aim of this study was to assess these reactions using FDG-PET and high-throughput methods such as gene profiling. We established Morris hepatoma (MH3924A) cell lines expressing TROP, sFLT or ASTAT and quantified 18F-fluorodeoxyglucose (18FDG) uptake by dynamic positron emission tomography (PET) after tumour inoculation in ACI rats. Furthermore, expression of glucose transporter-1 and -3 (GLUT-1 and GLUT-3) as well as hexokinase-1 and -2 were investigated by RT-PCR and immunohistomorphometry. In addition, gene array analyses were performed. 18FDG uptake, vascular fraction and distribution volume were significantly higher in all genetically modified tumours. Immunohistomorphometry showed an increased percentage of hexokinase-1 and -2 as well as GLUT-1 and -3 immunoreactive (ir) cells. Using gene arrays and comparing all three groups of genetically modified tumours, we found upregulated expression of 36 genes related to apoptosis, signal transduction, stress or metabolism. TROP-, sFLT- or ASTAT-expressing MH3924A tumours show enhanced influx of 18FDG, which seems to be caused by several factors: enhanced exchange of nutrients between blood and tumour, increased amounts of glucose transporters and hexokinases, and increased expression of genes related to apoptosis, matrix and stress, which induce an increased demand for glucose. (orig.)

  10. Stable transformation of moth bean Vigna aconitifolia via direct gene transfer.

    Science.gov (United States)

    Köhler, F; Golz, C; Eapen, S; Kohn, H; Schieder, O

    1987-07-01

    Direct gene transfer proved to be an efficient transformation method for Vigna aconitifolia, a member of the legume family. Kanamycin resistant calli and plants were regenerated from heat shocked protoplasts treated with PEG and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II). The plant cultivar used was an important factor in attaining higher transformation frequencies. Transformation was confirmed by Southern blot analysis using a non-radioactive detection system. Attempts to transform mesophyll and suspension cultured cells by this method were unsuccessful. Protoplasts electroporated with the plasmid pCAP212, which codes for chloramphenicol acetyltransferase, exhibited transient expression of this gene two days after treatment while electroporated cells did not show this enzyme activity. It is therefore assumed that the DNA uptake is prevented by the cell wall.

  11. Detecting horizontally transferred and essential genes based on dinucleotide relative abundance.

    Science.gov (United States)

    Baran, Robert H; Ko, Hanseok

    2008-10-01

    Various methods have been developed to detect horizontal gene transfer in bacteria, based on anomalous nucleotide composition, assuming that compositional features undergo amelioration in the host genome. Evolutionary theory predicts the inevitability of false positives when essential sequences are strongly conserved. Foreign genes could become more detectable on the basis of their higher order compositions if such features ameliorate more rapidly and uniformly than lower order features. This possibility is tested by comparing the heterogeneities of bacterial genomes with respect to strand-independent first- and second-order features, (i) G + C content and (ii) dinucleotide relative abundance, in 1 kb segments. Although statistical analysis confirms that (ii) is less inhomogeneous than (i) in all 12 species examined, extreme anomalies with respect to (ii) in the Escherichia coli K12 genome are typically co-located with essential genes.

  12. Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

    Science.gov (United States)

    Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

    2015-03-01

    Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

  13. Recombined adenovirus mediated delivery of p21 inhibits oxygen-induced retinal neovascularization in mice%重组腺病毒介导p21对小鼠视网膜新生血管生成的抑制作用

    Institute of Scientific and Technical Information of China (English)

    韩金栋; 袁志刚; 郑华宾; 颜华

    2012-01-01

    Objective To observe the the inhibitory effect of recombined adenovirus mediated delivery of p21 (rAd-p21) on oxygen-induced retinal neovascularization in mice.Methods A total of 56 C57BL/6 mice at the age of seven days were divided into control group,phosphate buffer solution (PBS) group,rAdp21 group and rAd-no purpose gene control (rAd-NC) group,14 mice in each group.The retinal neovascularization of PBS,rAd-p21and rAd-NC group were induced by oxygen,and received an intravitreal injection 1 μl PBS,rAd-p21 and rAd-NC at postnatal day 11,respectively.The rats of control group were not intervened.At postnatal day 17,RNV was determined by retinal flat mounts and retinal section; nonperfusion areas of retina were analyzed by Image-Pro plus 6.0 software; reverse transcription-polymerase chain reaction (RT-PCR) and Western blot was used to measure the mRNA and protein expression of p21 and CDK2.Results Compared with PBS and rAd-NC groups,the retinal non-perfusion areas,neovascularization and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in rAd-p21 group were reduced significantly.Non-perfusion areas of retina in rAd-p21 group was less than that in PBS and rAd-NC groups,the difference among these three groups was significantly (F= 101.634,P<0.05).Compared with the other three groups,the level of p21 mRNA and protein in rAd-p21 group increased significantly (F=839.664,509.817; P<0.05) ; the level of CDK2 mRNA and protein in rAd-p21 group decreased significantly (F=301.858,592.882; P<0.05).Conclusion rAd-p21can inhibit oxygen-induced retinal neovascularization,up-regulated p21 expression and down-regulated CDK2 expression may be the mechanism.%目的 观察重组腺病毒-p21 (rAd-p21)对氧诱导小鼠视网膜新生血管(RNV)的抑制作用.方法 将56只健康7日龄C57BL/6J小鼠随机分为对照组、磷酸盐缓冲液(PBS)组、rAd-p21组及rAd-无目的基因对照(rAd-NC)组,每组14只.PBS组、rAd-p21组及rAd

  14. Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer?

    Science.gov (United States)

    Tormo, M Angeles; Knecht, Erwin; Götz, Friedrich; Lasa, Iñigo; Penadés, José R

    2005-07-01

    The biofilm-associated protein (Bap) is a surface protein implicated in biofilm formation by Staphylococcus aureus isolated from chronic mastitis infections. The bap gene is carried in a putative composite transposon inserted in SaPIbov2, a mobile staphylococcal pathogenicity island. In this study, bap orthologue genes from several staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus, were identified, cloned and sequenced. Sequence analysis comparison of the bap gene from these species revealed a very high sequence similarity, suggesting the horizontal gene transfer of SaPIbov2 amongst them. However, sequence analyses of the flanking region revealed that the bap gene of these species was not contained in the SaPIbov2 pathogenicity island. Although they did not contain the icaADBC operon, all the coagulase-negative staphylococcal isolates harbouring bap were strong biofilm producers. Disruption of the bap gene in S. epidermidis abolished its capacity to form a biofilm, whereas heterologous complementation of a biofilm-negative strain of S. aureus with the Bap protein from S. epidermidis bestowed the capacity to form a biofilm on a polystyrene surface. Altogether, these results demonstrate that Bap orthologues from coagulase-negative staphylococci induce an alternative mechanism of biofilm formation that is independent of the PIA/PNAG exopolysaccharide.

  15. Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer.

    Science.gov (United States)

    Richard-Fiardo, Peggy; Hervouet, Catherine; Marsault, Robert; Franken, Philippe R; Cambien, Béatrice; Guglielmi, Julien; Warnez-Soulie, Julie; Darcourt, Jacques; Pourcher, Thierry; Colombani, Thibault; Haudebourg, Thomas; Peuziat, Pauline; Pitard, Bruno; Vassaux, Georges

    2015-03-01

    In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs. PMID:25662490

  16. The advantages and disadvantages of horizontal gene transfer and the emergence of the first species

    Directory of Open Access Journals (Sweden)

    Higgs Paul G

    2011-01-01

    Full Text Available Abstract Background Horizontal Gene Transfer (HGT is beneficial to a cell if the acquired gene confers a useful function, but is detrimental if the gene has no function, if it is incompatible with existing genes, or if it is a selfishly replicating mobile element. If the balance of these effects is beneficial on average, we would expect cells to evolve high rates of acceptance of horizontally transferred genes, whereas if it is detrimental, cells should reduce the rate of HGT as far as possible. It has been proposed that the rate of HGT was very high in the early stages of prokaryotic evolution, and hence there were no separate lineages of organisms. Only when the HGT rate began to fall, would lineages begin to emerge with their own distinct sets of genes. Evolution would then become more tree-like. This phenomenon has been called the Darwinian Threshold. Results We study a model for genome evolution that incorporates both beneficial and detrimental effects of HGT. We show that if rate of gene loss during genome replication is high, as was probably the case in the earliest genomes before the time of the last universal common ancestor, then a high rate of HGT is favourable. HGT leads to the rapid spread of new genes and allows the build-up of larger, fitter genomes than could be achieved by purely vertical inheritance. In contrast, if the gene loss rate is lower, as in modern prokaryotes, then HGT is, on average, unfavourable. Conclusions Modern cells should therefore evolve to reduce HGT if they can, although the prevalence of independently replicating mobile elements and viruses may mean that cells cannot avoid HGT in practice. In the model, natural selection leads to gradual improvement of the replication accuracy and gradual decrease in the optimal rate of HGT. By clustering genomes based on gene content, we show that there are no separate lineages of organisms when the rate of HGT is high; however, as the rate of HGT decreases, a tree

  17. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer

    Institute of Scientific and Technical Information of China (English)

    TomohikoICHIKAWA; ShigeruHOSOKI; HiroyoshiSUZUKI; KoichiroAKAKURA; TatsuoIGARASHI; YuzoFURUYA; MitsuoOSHIMURA; CarrieW.RINKER-SCHAEFFER; NaokiNIHEI; JohnT.ISAACS; HaruoITO

    2000-01-01

    Aim: To identify the metastasis suppressor genes for prostate cancer. Methods: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene (s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. Results: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of ttmaorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal ann deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. Conclusion: This assay system is useful to identify metastasis suppressor gene (s) for prostate cancer.

  18. Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection

    OpenAIRE

    Ulf Sorhannus

    2012-01-01

    I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among “distantly” related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus—Hemitripterus americanus clade to the ancestor of the Hypomesus ...

  19. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    Science.gov (United States)

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C

    1998-01-01

    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  20. Contribution of Multiple Inter-Kingdom Horizontal Gene Transfers to Evolution and Adaptation of Amphibian-Killing Chytrid, Batrachochytrium dendrobatidis

    Science.gov (United States)

    Sun, Baofa; Li, Tong; Xiao, Jinhua; Liu, Li; Zhang, Peng; Murphy, Robert W.; He, Shunmin; Huang, Dawei

    2016-01-01

    Amphibian populations are experiencing catastrophic declines driven by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although horizontal gene transfer (HGT) facilitates the evolution and adaptation in many fungi by conferring novel function genes to the recipient fungi, inter-kingdom HGT in Bd remains largely unexplored. In this study, our investigation detects 19 bacterial genes transferred to Bd, including metallo-beta-lactamase and arsenate reductase that play important roles in the resistance to antibiotics and arsenates. Moreover, three probable HGT gene families in Bd are from plants and one gene family coding the ankyrin repeat-containing protein appears to come from oomycetes. The observed multi-copy gene families associated with HGT are probably due to the independent transfer events or gene duplications. Five HGT genes with extracellular locations may relate to infection, and some other genes may participate in a variety of metabolic pathways, and in doing so add important metabolic traits to the recipient. The evolutionary analysis indicates that all the transferred genes evolved under purifying selection, suggesting that their functions in Bd are similar to those of the donors. Collectively, our results indicate that HGT from diverse donors may be an important evolutionary driver of Bd, and improve its adaptations for infecting and colonizing host amphibians.

  1. Contribution of Multiple Inter-kingdom Horizontal Gene Transfers to Evolution and Adaptation of Amphibian-killing Chytrid, Batrachochytrium dendrobatidis

    Directory of Open Access Journals (Sweden)

    Baofa Sun

    2016-08-01

    Full Text Available Amphibian populations are experiencing catastrophic declines driven by the fungal pathogen Batrachochytrium dendrobatidis (Bd. Although horizontal gene transfer (HGT facilitates the evolution and adaptation in many fungi by conferring novel function genes to the recipient fungi, inter-kingdom HGT in Bd remains largely unexplored. In this study, our investigation detects 19 bacterial genes transferred to Bd, including metallo-beta-lactamase and arsenate reductase that play important roles in the resistance to antibiotics and arsenates. Moreover, three probable HGT gene families in Bd are from plants and one gene family coding the ankyrin repeat-containing protein appears to come from oomycetes. The observed multi-copy gene families associated with HGT are probably due to the independent transfer events or gene duplications. Five HGT genes with extracellular locations may relate to infection, and some other genes may participate in a variety of metabolic pathways, and in doing so add important metabolic traits to the recipient. The evolutionary analysis indicates that all the transferred genes evolved under purifying selection, suggesting that their functions in Bd are similar to those of the donors. Collectively, our results indicate that HGT from diverse donors may be an important evolutionary driver of Bd, and improve its adaptations for infecting and colonizing host amphibians.

  2. Contribution of Multiple Inter-Kingdom Horizontal Gene Transfers to Evolution and Adaptation of Amphibian-Killing Chytrid, Batrachochytrium dendrobatidis

    Science.gov (United States)

    Sun, Baofa; Li, Tong; Xiao, Jinhua; Liu, Li; Zhang, Peng; Murphy, Robert W.; He, Shunmin; Huang, Dawei

    2016-01-01

    Amphibian populations are experiencing catastrophic declines driven by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although horizontal gene transfer (HGT) facilitates the evolution and adaptation in many fungi by conferring novel function genes to the recipient fungi, inter-kingdom HGT in Bd remains largely unexplored. In this study, our investigation detects 19 bacterial genes transferred to Bd, including metallo-beta-lactamase and arsenate reductase that play important roles in the resistance to antibiotics and arsenates. Moreover, three probable HGT gene families in Bd are from plants and one gene family coding the ankyrin repeat-containing protein appears to come from oomycetes. The observed multi-copy gene families associated with HGT are probably due to the independent transfer events or gene duplications. Five HGT genes with extracellular locations may relate to infection, and some other genes may participate in a variety of metabolic pathways, and in doing so add important metabolic traits to the recipient. The evolutionary analysis indicates that all the transferred genes evolved under purifying selection, suggesting that their functions in Bd are similar to those of the donors. Collectively, our results indicate that HGT from diverse donors may be an important evolutionary driver of Bd, and improve its adaptations for infecting and colonizing host amphibians. PMID:27630622

  3. Fat-to-glucose interconversion by hydrodynamic transfer of two glyoxylate cycle enzyme genes

    Directory of Open Access Journals (Sweden)

    Marzo F

    2008-12-01

    Full Text Available Abstract The glyoxylate cycle, which is well characterized in higher plants and some microorganisms but not in vertebrates, is able to bypass the citric acid cycle to achieve fat-to-carbohydrate interconversion. In this context, the hydrodynamic transfer of two glyoxylate cycle enzymes, such as isocytrate lyase (ICL and malate synthase (MS, could accomplish the shift of using fat for the synthesis of glucose. Therefore, 20 mice weighing 23.37 ± 0.96 g were hydrodinamically gene transferred by administering into the tail vein a bolus with ICL and MS. After 36 hours, body weight, plasma glucose, respiratory quotient and energy expenditure were measured. The respiratory quotient was increased by gene transfer, which suggests that a higher carbohydrate/lipid ratio is oxidized in such animals. This application could help, if adequate protocols are designed, to induce fat utilization for glucose synthesis, which might be eventually useful to reduce body fat depots in situations of obesity and diabetes.

  4. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    Directory of Open Access Journals (Sweden)

    Shih Ping Yao

    2002-04-01

    Full Text Available Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C, is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57 of transgenic pigs (F0 generation. Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

  5. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

    Directory of Open Access Journals (Sweden)

    Luthey-Schulten Zaida

    2009-07-01

    Full Text Available Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding and C- (zinc-free variants, with 26 genomes (mainly from the class Clostridia containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution

  6. Different fates of the chloroplast tufA gene following its transfer to the nucleus in green algae.

    OpenAIRE

    Baldauf, S L; Manhart, J R; J.D. Palmer

    1990-01-01

    Previous work suggested that the tufA gene, encoding protein synthesis elongation factor Tu, was transferred from the chloroplast to the nucleus within the green algal lineage giving rise to land plants. In this report we investigate the timing and mode of transfer by examining chloroplast and nuclear DNA from the three major classes of green algae, with emphasis on the class Charophyceae, the proposed sister group to land plants. Filter hybridizations reveal a chloroplast tufA gene in all Ul...

  7. Microsporidia: Eukaryotic Intracellular Parasites Shaped by Gene Loss and Horizontal Gene Transfers.

    Science.gov (United States)

    Corradi, Nicolas

    2015-01-01

    Microsporidia are eukaryotic parasites of many animals that appear to have adapted to an obligate intracellular lifestyle by modifying the morphology and content of their cells. Living inside other cells, they have lost many, or all, metabolic functions, resulting in genomes that are always gene poor and often very small. The minute content of microsporidian genomes led many to assume that these parasites are biochemically static and uninteresting. However, recent studies have demonstrated that these organisms can be surprisingly complex and dynamic. In this review I detail the most significant recent advances in microsporidian genomics and discuss how these have affected our understanding of many biological aspects of these peculiar eukaryotic intracellular pathogens. PMID:26195306

  8. Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters

    Science.gov (United States)

    Miller, Jennifer H.; Novak, John T.; Knocke, William R.; Pruden, Amy

    2016-01-01

    Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1—a Pseudomonas sp.) and thermophilic (Iso T10—a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457–0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130–0.486, P = 0.075–0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and

  9. Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters.

    Science.gov (United States)

    Miller, Jennifer H; Novak, John T; Knocke, William R; Pruden, Amy

    2016-01-01

    Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1-a Pseudomonas sp.) and thermophilic (Iso T10-a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457-0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130-0.486, P = 0.075-0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and/or horizontal gene

  10. Algal endosymbionts as vectors of horizontal gene transfer in photosynthetic eukaryotes

    Directory of Open Access Journals (Sweden)

    Huan eQiu

    2013-09-01

    Full Text Available Photosynthesis in eukaryotes occurs in the plastid, an organelle that is derived from a single cyanobacterial primary endosymbiosis in the common ancestor of the supergroup Plantae (or Archaeplastida that includes green, red, and glaucophyte algae and plants. However a variety of other phytoplankton such as the chlorophyll c-containing diatoms, dinoflagellates, and haptophytes contain a red alga-derived plastid that traces its origin to secondary or tertiary (eukaryote engulfs eukaryote endosymbiosis. The hypothesis of Plantae monophyly has only recently been substantiated, however the extent and role of endosymbiotic and horizontal gene transfer (EGT and HGT in algal genome evolution still remain to be fully understood. What is becoming clear from analysis of complete genome data is that algal gene complements can no longer be considered essentially eukaryotic in provenance; i.e., with the expected addition of several hundred cyanobacterial genes derived from EGT and a similar number derived from the mitochondrial ancestor. For example, we now know that foreign cells such as Chlamydiae and other prokaryotes have made significant contributions to plastid functions in Plantae. Perhaps more surprising is the recent finding of extensive bacterium-derived HGT in the nuclear genome of the unicellular red alga Porphyridium purpureum that does not relate to plastid functions. These non-endosymbiont gene transfers not only shaped the evolutionary history of Plantae but also were propagated via secondary endosymbiosis to a multitude of other phytoplankton. Here we discuss the idea that Plantae (in particular red algae are one of the major players in eukaryote genome evolution by virtue of their ability to act as sinks and sources of foreign genes through HGT and endosymbiosis, respectively. This hypothesis recognizes the often under-appreciated Rhodophyta as major sources of genetic novelty among photosynthetic eukaryotes.

  11. Transfer of genes for stem rust resistance from Agropyron elongatum and imperial rye to durum wheat

    International Nuclear Information System (INIS)

    The Agropyron elongatum gene for stem rust resistance on chromosome 6A of Knott's Thatcher translocation line was transferred to a susceptible local durum wheat variety, Jaya, through a series of back-crosses. Plants heterozygous for the Agropyron translocation always show at least one open bivalent. Homozygotes have not been obtained, probably because of the absence of male transmission in durum background. Monotelosomic addition of the short arm of Imperial rye chromosome 3R (formerly ''G'' of Sears), which carries a gene(s) for resistance to wheat stem rust, was obtained in the local durum variety. Rust-resistant plants from parents having the added rye telocentric were irradiated with gamma rays just before meiosis, and the pollen obtained from the irradiated spikes was used to pollinate euploid plants. In addition, seeds harvested from 2n+1 resistant plants were irradiated with thermal neutrons and the resistant M1 plants were selfed to raise M2 families. Two durum-rye translocation lines were obtained following irradiation. DRT-1 was transmitted normally through the female gametes but showed no male transmission. As a result of this, homozygotes have not been obtained. Gametic transmission rates of DRT-2 are being tested. Alien translocations, which show normal gametic and zygotic transmissions in the hexaploid wheat, may behave differently in a tetraploid background. The results indicate that alien genetic transfers may be more difficult to obtain in durum wheat, probably owing to the reduced buffering effect of the tetraploid genome. (author)

  12. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in vivo: Polyethylenimine

    Science.gov (United States)

    Boussif, Otmane; Lezoualc'h, Frank; Zanta, Maria Antonietta; Djavaheri Mergny, Mojgan; Scherman, Daniel; Demeneix, Barbara; Behr, Jean-Paul

    1995-08-01

    Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se-i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its genedelivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

  13. Insulin mediated hemodynamic responses in spontaneous hypertensive rats (SHRs): effect of chromosome 4 gene transfer.

    Science.gov (United States)

    Rao, Sumangala P; McRae, Crystal; Lapanowski, Karen; Churchill, Monique; Kurtz, Theodore W; Dunbar, Joseph C

    2003-02-01

    The spontaneous hypertensive rat (SHR) is a widely studied model of essential hypertension and has been reported to exhibit alterations in carbohydrate and lipid metabolism. Genetic linkage studies implicated that SHR carries deletion variant of Cd36 gene of chromosome 4, the gene that encodes fatty acid transporter. Thus it could be possible that primary genetic defect in SHR is compromised tissue utilization of fatty acid that would form the basis for the pathogenesis of hyperinsulinemia, insulin resistance and insulin-mediated responses. We measured both the hemodynamic and metabolic responses to insulin in SHR in comparison with the chromosome congenic spontaneous hypertensive rats (cSHRs) (rats in which piece of chromosome 4 containing wild type Cd36 was integrated into the SHR genome). A bolus infusion of insulin increased iliac conductance and decreased blood pressure in Wistar Kyoto (WKY) rats. However, in SHR insulin did not reduce blood pressure as in WKY but after about 15 min it significantly enhanced blood pressure and reduced iliac conductance. Whereas in cSHR insulin did not reduce blood pressure as in WKY rats. However, pressor responses to insulin were eliminated by chromosome 4 gene transfer. Glucose clearance was significantly slower in both SHR and cSHR. Glucose tolerance test revealed that SHR are hyperinsulinemic and insulin resistant. These findings indicate that transfer of segment of chromosome 4 from Brown Norway rats onto spontaneous hypertensive background eliminates hyperinsulinemia and pressor effects of insulin.

  14. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice.

    Science.gov (United States)

    Heckmann, M B; Bauer, R; Jungmann, A; Winter, L; Rapti, K; Strucksberg, K-H; Clemen, C S; Li, Z; Schröder, R; Katus, H A; Müller, O J

    2016-08-01

    Mutations of the human desmin (DES) gene cause autosomal dominant and recessive myopathies affecting skeletal and cardiac muscle tissue. Desmin knockout mice (DES-KO), which develop progressive myopathy and cardiomyopathy, mirror rare human recessive desminopathies in which mutations on both DES alleles lead to a complete ablation of desmin protein expression. Here, we investigated whether an adeno-associated virus-mediated gene transfer of wild-type desmin cDNA (AAV-DES) attenuates cardiomyopathy in these mice. Our approach leads to a partial reconstitution of desmin protein expression and the de novo formation of the extrasarcomeric desmin-syncoilin network in cardiomyocytes of treated animals. This finding was accompanied by reduced fibrosis and heart weights and improved systolic left-ventricular function when compared with control vector-treated DES-KO mice. Since the re-expression of desmin protein in cardiomyocytes of DES-KO mice restores the extrasarcomeric desmin-syncoilin cytoskeleton, attenuates the degree of cardiac hypertrophy and fibrosis, and improves contractile function, AAV-mediated desmin gene transfer may be a novel and promising therapeutic approach for patients with cardiomyopathy due to the complete lack of desmin protein expression. PMID:27101257

  15. Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

    Directory of Open Access Journals (Sweden)

    Oksana V Arkhipova

    Full Text Available The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd and tetraheme cytochrome с (Mcc in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.

  16. Transfer of Lysozyme Gene into indica Parents of Hybrid Rice by Backcrossing

    Institute of Scientific and Technical Information of China (English)

    YI Zi-li; WANG Zi-xuan; QIN Jing-ping; JIANG Jian-xiong; TAN Yan-ning; ZHOU Qing-ming

    2006-01-01

    Alysozyme gene resistant to rice blast was transferred from the donor transgenic japonica rice Zhonghua 9 (D2-1-2) into a sterile line Pei'ai 64S(PA 64S) and restorer line 9311 of the two-line hybrid rice Liangyoupeijiu, and the restorer line Minghui 63 (MH63) of three-line hybrid rice Shanyou 63 by successive backcrossing. The PCR analysis confirmed that foreign lysozyme gene was B2F2 9311, B2F2 MH63 and B1F2 PA64S, indicating that the foreign gene was stably inherited over successive generations as a dominant single copy gene. The resistance against rice blast in backcross or selfed generations and corresponding testcross combinations were investigated in 2003 and 2004. The results showed that the resistance of the transgenic rice to blast had a greater improvement than that of the corresponding recurrent parents or the corresponding check hybrid combinations. The resistance of the advanced backcross and selfed generations to rice blast is much stronger than that of the early generations. The study confirmed thattransferring the lysozyme gene into hybrid parents by backcrossing was a simple and effective approach to develop new hybrid rice resistant to rice blast.

  17. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    International Nuclear Information System (INIS)

    Highlights: → A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. → The promoter was characterized with radiation-inducibility and tumor-specificity. → Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. → Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  18. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  19. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    孙永华; 陈尚萍; 汪亚平; 朱作言

    2000-01-01

    The transcriptional onset ot hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

  20. The onset of foreign gene transcription in nuclear-transferred embryos of fish

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blas-tula stage and even 16-cell stage in the first, second and third cases, respectively. It looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

  1. Adaptive eukaryote-to-eukaryote lateral gene transfer: stress-related genes of algal origin in the closest unicellular relatives of animals.

    Science.gov (United States)

    Nedelcu, A M; Miles, I H; Fagir, A M; Karol, K

    2008-11-01

    In addition to mutation, gene duplication and recombination, the transfer of genetic material between unrelated species is now regarded as a potentially significant player in the shaping of extant genomes and the evolution and diversification of life. Although this is probably true for prokaryotes, the extent of such genetic exchanges in eukaryotes (especially eukaryote-to-eukaryote transfers) is more controversial and the selective advantage and evolutionary impact of such events are less documented. A laterally transferred gene could either be added to the gene complement of the recipient or replace the recipient's homologue; whereas gene replacements can be either adaptive or stochastic, gene additions are most likely adaptive. Here, we report the finding of four stress-related genes (two ascorbate peroxidase and two metacaspase genes) of algal origin in the closest unicellular relatives of animals, the choanoflagellates. At least three of these sequences represent additions to the choanoflagellate gene complement, which is consistent with these transfers being adaptive. We suggest that these laterally acquired sequences could have provided the primitive choanoflagellates with additional or more efficient means to cope with stress, especially in relation to adapting to freshwater environments and/or sessile or colonial lifestyles. PMID:18717747

  2. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    Science.gov (United States)

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism. PMID:23800623

  3. Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

    Science.gov (United States)

    Nomikou, Nikolitsa; Feichtinger, Georg A; Redl, Heinz; McHale, Anthony P

    2016-01-01

    It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration.

  4. Transient hypermutagenesis accelerates the evolution of legume endosymbionts following horizontal gene transfer.

    Directory of Open Access Journals (Sweden)

    Philippe Remigi

    2014-09-01

    Full Text Available Horizontal gene transfer (HGT is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and β-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT.

  5. Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling.

    Science.gov (United States)

    Liu, Cun-Fei; Zhang, Jia; Shen, Kai; Gao, Ping-Jin; Wang, Hai-Ya; Jin, Xin; Meng, Chao; Fang, Ning-Yuan

    2015-04-01

    Vascular adventitia and adventitia‑derived reactive oxygen species (ROS) contribute to vascular remodeling following vascular injury. A previous ex vivo study in adventitial fibroblasts showed that catalase, one of most important anti‑oxide enzymes, was downregulated by angiotensin II (AngII). The aim of the present study was to investigate whether adventitial gene transfer of catalase affects AngII‑induced vascular remodeling in vivo. Adenoviruses co‑expressing catalase and enhanced green fluorescent protein (eGFP) or expressing eGFP only were applied to the adventitial surface of common carotid arteries of Sprague‑Dawley rats. Alzet minipumps administering AngII (0.75 mg/kg/day) were then implanted subcutaneously for 14 days. Systolic blood pressure and biological parameters of vascular remodeling were measured in each group. Adventitial fibroblasts were cultured and p38 mitogen‑activated protein kinase (MAPK) phosphorylation was measured using western blot analysis. The results showed that adventitial gene transfer of catalase had no effect on AngII‑induced systolic blood pressure elevation. However, catalase adenovirus transfection significantly inhibited AngII‑induced media hypertrophy compared with that of the control virus (Pcatalase transfection significantly attenuated AngII‑induced ROS generation, macrophage infiltration, collagen deposition and adventitial α‑smooth muscle actin expression. Furthermore, catalase transfection significantly inhibited the AngII‑induced increase in p38MAPK phosphorylation. In conclusion, the results of the present study demonstrated that adventitial gene transfer of catalase significantly attenuated AngII‑induced vascular remodeling in rats via inhibition of adventitial p38MAPK phosphorylation.

  6. Transcriptional regulation of pWW0 transfer genes in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    Lambertsen, L.M.; Molin, Søren; Kroer, N.;

    2004-01-01

    of whose gene products are predicted to be involved in relaxasome formation and DNA processing during transfer, and they are repressed by TraA. The third promoter region, upstream of mpfR, is responsible for transcription of mpfR and mpfA to mpfJ, encoding proteins involved in mating pair formation....... This suggests that the general pattern of genetic organisation exhibited by these systems has arisen a number of times independently and must therefore be highly favourable to plasmid survival and spread....

  7. Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections

    DEFF Research Database (Denmark)

    GLUD, AN; Hedegaard, Claus; nielsen, MS;

    2010-01-01

    We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6...... per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin...

  8. Horizontal gene transfer and redundancy of tryptophan biosynthetic enzymes in dinotoms.

    Science.gov (United States)

    Imanian, Behzad; Keeling, Patrick J

    2014-02-01

    A tertiary endosymbiosis between a dinoflagellate host and diatom endosymbiont gave rise to "dinotoms," cells with a unique nuclear and mitochondrial redundancy derived from two evolutionarily distinct eukaryotic lineages. To examine how this unique redundancy might have affected the evolution of metabolic systems, we investigated the transcription of genes involved in biosynthesis of the amino acid tryptophan in three species, Durinskia baltica, Kryptoperidinium foliaceum, and Glenodinium foliaceum. From transcriptome sequence data, we recovered two distinct sets of protein-coding transcripts covering the entire tryptophan biosynthetic pathway. Phylogenetic analyses suggest a diatom origin for one set of the proteins, which we infer to be expressed in the endosymbiont, and that the other arose from multiple horizontal gene transfer events to the dinoflagellate ancestor of the host lineage. This is the first indication that these cells retain redundant sets of transcripts and likely metabolic pathways for the biosynthesis of small molecules and extend their redundancy to their two distinct nuclear genomes. PMID:24448981

  9. VERTICAL HEREDITY VS. HORIZONTAL GENE TRANSFER: A CHALLENGE TO BACTERIAL CLASSIFICATION

    Institute of Scientific and Technical Information of China (English)

    HAO Bailin; QI Ji

    2003-01-01

    The diversity and classification of microbes has been a long-standing issue. Molecular phylogeny of the prokaryotes based on comparison of the 16S rRNA sequences of the small ribosomal subunit has led to a reasonable tree of life in the late 1970s. However, the availability of more and more complete bacterial genomes has brought about complications instead of refinement of the tree. In particular, it turns out that different choice of genes may tell different history. This might be caused by possible horizontal gene transfer (HGT) among species. There is an urgent need to develop phylogenetic methods that make use of whole genome data. We describe a new approach in molecular phylogeny, namely, tree construction based on K-tuple frequency analysis of the genomic sequences. Putting aside the technicalities, we emphasize the transition from randomness to determinism when the string length K increases and try to comment on the challenge mentioned in the title.

  10. Localized gene transfer into organotypic hippocampal slice cultures and acute hippocampal slices

    DEFF Research Database (Denmark)

    Casaccia-Bonnefil, P; Benedikz, Eirikur; Shen, H;

    1993-01-01

    Viral vectors derived from herpes simplex virus, type-1 (HSV), can transfer and express genes into fully differentiated, post-mitotic neurons. These vectors also transduce cells effectively in organotypic hippocampal slice cultures. Nanoliter quantities of a virus stock of HSVlac, an HSV vector...... effective and rapid. The titer of the HSVlac stocks was determined on NIH3T3 cells. Eighty-three percent of the beta-gal forming units successfully transduced beta-gal after microapplication to slice cultures. beta-Gal expression was detected as rapidly as 4 h after transduction into cultures of fibroblasts...... or hippocampal slices. The rapid expression of beta-gal by HSVlac allowed efficient transduction of acute hippocampal slices. Many genes have been transduced and expressed using HSV vectors; therefore, this microapplication method can be applied to many neurobiological questions....

  11. Pharmacokinetics and in vivo gene transfer of plasmid DNA complexed with mannosylated poly(L-lysine) in mice

    NARCIS (Netherlands)

    Nishikawa, M; Takemura, S; Yamashita, F; Takakura, Y; Meijer, DKF; Hashida, M; Swart, PJ

    2000-01-01

    To achieve mannose receptor-mediated, cell-specific, in vivo gene transfer by intravenous injection of plasmid DNA, mannosylated poly(l-lysine) (Man-PLL) was synthesized as a carrier molecule, and mixed with a plasmid DNA encoding chloramphenicol acetyltransferase (CAT) gene to form DNA/Man-PLL comp

  12. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  13. Transfer and expression of the gene for human growth hormone in Oryza sativa and Phleum pratense L

    International Nuclear Information System (INIS)

    Two derived pB322 plasmids harboring bla (for Beta-lactamase genes) and Human growth Hormone (hGH) genes, were transferred to a cereal plant (Rice) and to a gramine ous fodder (Timothy) in which they are expressed, whereas those constructions are not expressed in Escherichia coli.

  14. Induced differentiation of C2C12 to osteoblast via adenovirus-mediated Cbfa1 in vitro%体外诱导C2C12细胞向成骨细胞的分化

    Institute of Scientific and Technical Information of China (English)

    张勇; 杨彤涛; 胡运生; 廖博; 文艳华; 范清宇

    2013-01-01

    目的 成骨细胞特异性转录因子a1(core binding factor a1,Cbfa1)通过调节生长因子和骨特异性细胞外基质蛋白的基因表达而参与成骨细胞的分化和骨发育过程.文中构建成Cbfa1,以腺病毒载体转染成肌细胞C2C12,为种子细胞构建组织工程化骨.方法 体外培养小鼠成肌细胞C2C12,用重组腺病毒质粒pAd-IL-31介导Cbfa1/Osf2基因瞬时转染小鼠成肌C2C12细胞,Western blot检测Cbfa1蛋白表达.结果 Cbfa1蛋白表达、碱性磷酸酶(alkaline phosphatase,ALP)活性测定、骨钙素(osteocalcin,OCN)分泌量以及茜素红染色感染组明显高于对照组.结论 成肌细胞C2C12可以作为种子细胞构建组织工程化骨.%Objective Osteoblast core binding factor a 1 ( Cbfal) plays a role in osteoblast differentiation and development by regulating the gene of growth factor and extracellular matrix proteins . Recombinant adenovirus vector mediated Cbfa 1 was transferred to myoblast C2C12 to construct the tissue-engineered bone. Methods The myoblast C2C12 was cultured in vitro, and then transiently transfected with recombinant adenovirus vector pAd -IL-31 mediated-Cbfal/Osf2. Western blot was used to detect the expression of Cbfal. Results Compared with the control group , the expression of Cbfal, activity of alkaline phosphtase (ALP) , secretory volume of osteocalcin (OCN) and staining via alizarin bordeaux were higher in the transfection group . Conclusion Myoblast C2C12 acts as a seed cell for constructing tissue -engineered bone.

  15. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

    Science.gov (United States)

    Xiao, Fei; Deng, Jiali; Yu, Junjie; Guo, Yajie; Chen, Shanghai; Guo, Feifan

    2016-01-01

    Insulin resistance is one of the major factors contributing to metabolic diseases, but the underlying mechanisms are still poorly understood. As an important cofactor, B-cell translocation gene 1 (BTG1) is involved in many physiologic processes; however, the direct effect of BTG1 on insulin sensitivity has not been described. In our study, BTG1 overexpression or knockdown improved or impaired insulin signaling in vitro, respectively. In addition, adenovirus-mediated BTG1 overexpression improved insulin sensitivity in wild-type (WT) and insulin-resistant leptin-receptor mutated (db/db) mice. In addition, transgenic BTG1-overexpressing mice were resistant to high-carbohydrate diet-induced insulin resistance. Adenovirus-mediated BTG1 knockdown consistently impaired insulin sensitivity in WT and insulin-sensitive leucine-deprived mice. Moreover, hepatic BTG1 expression was increased by leucine deprivation via the mammalian target of rapamycin/ribosomal protein S6 kinase 1 pathway. Furthermore, c-Jun expression was up-regulated by BTG1, and adenovirus-mediated c-Jun knockdown blocked BTG1-improved insulin signaling and insulin sensitivity in vitro and in vivo. Finally, BTG1 promoted c-Jun expression via stimulating c-Jun and retinoic acid receptor activities. Taken together, these results identify a novel function for BTG1 in the regulation of hepatic insulin sensitivity and provide important insights into the nutritional regulation of BTG1 expression.- Xiao, F., Deng, J., Yu, J., Guo, Y., Chen, S., Guo, F. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

  16. Iodide uptake in human anaplastic thyroid carcinoma cells after transfer of the human thyroid peroxidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Haberkom, U. [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Dept. of Nuclear Medicine, Univ. of Heidelberg (Germany); Altmann, A.; Jiang, S.; Morr, I.; Mahmut, M. [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Eisenhut, M. [Dept. of Nuclear Medicine, Univ. of Heidelberg (Germany)

    2001-05-01

    Human thyroperoxidase (hTPO) is critical for the accumulation of iodide in thyroid tissues. Poorly differentiated and anaplastic thyroid tumours which lack thyroid-specific gene expression fail to accumulate iodide and, therefore, do not respond to iodine-131 therapy. We consequently investigated whether transfer of the hTPO gene is sufficient to restore the iodide-trapping capacity in undifferentiated thyroid and non-thyroid tumour cells. The human anaplastic thyroid carcinoma cell lines C643 and SW1736, the rat Morris hepatoma cell line MH3924A and the rat papillary thyroid carcinoma cell line L2 were used as in vitro model systems. Employing a bicistronic retroviral vector based on the myeloproliferative sarcoma virus for the transfer of the hTPO and the neomycin resistance gene, the C643 cells and SW1736 cells were transfected while the L2 cells and MH3924A cells were infected with retroviral particles. Seven recombinant C643 and seven SW1736 cell lines as well as four recombinant L2 and four MH3924A cell lines were established by neomycin selection. They were studied for hTPO expression using an antibody-based luminescence kit, followed by determination of the enzyme activity in the guaiacol assay and of the iodide uptake capacity in the presence of Na{sup 125}I. Genetically modified cell lines expressed up to 1,800 times more hTPO as compared to wild type tumour cells. The level of hTPO expression varied significantly between individual neomycin-resistant cell lines, suggesting that the recombinant retroviral DNA was integrated at different sites of the cellular genome. The accumulation of iodide, however, was not significantly enhanced in individual recombinant cell lines, irrespective of low or high hTPO expression. Moreover, there was no correlation between hTPO expression and enzyme activity in individual cell lines. The transduction of the hTPO gene per se is not sufficient to restore iodide trapping in non-iodide-concentrating tumour cells. Future

  17. Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria.

    Directory of Open Access Journals (Sweden)

    Shimon Bershtein

    2015-10-01

    Full Text Available Horizontal gene transfer (HGT plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR, with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10-90% in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM, correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the

  18. DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

    International Nuclear Information System (INIS)

    An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10-5 and 10-4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

  19. Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

    NARCIS (Netherlands)

    Haisma, Hidde J.; Kamps, Jan A. A. M.; Plantinga, Josee A.; Rots, Marianne G.; Bellu, Anna Rita; Kamps, Grietina

    2008-01-01

    Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the

  20. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

    Science.gov (United States)

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-07-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.

  1. Genetic diversity of bacterial communities and gene transfer agents in northern South China Sea.

    Directory of Open Access Journals (Sweden)

    Fu-Lin Sun

    Full Text Available Pyrosequencing of the 16S ribosomal RNA gene (rDNA amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA major capsid gene (g5 was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS, temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments.

  2. Design and bioinformatics analysis of novel biomimetic peptides as nanocarriers for gene transfer

    Directory of Open Access Journals (Sweden)

    Asia Majidi

    2015-01-01

    Full Text Available Objective(s: The introduction of nucleic acids into cells for therapeutic objectives is significantly hindered by the size and charge of these molecules and therefore requires efficient vectors that assist cellular uptake. For several years great efforts have been devoted to the study of development of recombinant vectors based on biological domains with potential applications in gene therapy. Such vectors have been synthesized in genetically engineered approach, resulting in biomacromolecules with new properties that are not present in nature. Materials and Methods: In this study, we have designed new peptides using homology modeling with the purpose of overcoming the cell barriers for successful gene delivery through Bioinformatics tools. Three different carriers were designed and one of those with better score through Bioinformatics tools was cloned, expressed and its affinity for pDNA was monitored. Results: The resultszz demonstrated that the vector can effectively condense pDNAinto nanoparticles with the average sizes about 100 nm. Conclusion: We hope these peptides can overcome the biological barriers associated with gene transfer, and mediate efficient gene delivery.

  3. A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents.

    Science.gov (United States)

    Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

    2016-01-01

    Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205-279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics. PMID:27460944

  4. Comparative genomics of mitochondria in chlorarachniophyte algae: endosymbiotic gene transfer and organellar genome dynamics

    Science.gov (United States)

    Tanifuji, Goro; Archibald, John M.; Hashimoto, Tetsuo

    2016-02-01

    Chlorarachniophyte algae possess four DNA-containing compartments per cell, the nucleus, mitochondrion, plastid and nucleomorph, the latter being a relic nucleus derived from a secondary endosymbiont. While the evolutionary dynamics of plastid and nucleomorph genomes have been investigated, a comparative investigation of mitochondrial genomes (mtDNAs) has not been carried out. We have sequenced the complete mtDNA of Lotharella oceanica and compared it to that of another chlorarachniophyte, Bigelowiella natans. The linear mtDNA of L. oceanica is 36.7 kbp in size and contains 35 protein genes, three rRNAs and 24 tRNAs. The codons GUG and UUG appear to be capable of acting as initiation codons in the chlorarachniophyte mtDNAs, in addition to AUG. Rpl16, rps4 and atp8 genes are missing in L.oceanica mtDNA, despite being present in B. natans mtDNA. We searched for, and found, mitochondrial rpl16 and rps4 genes with spliceosomal introns in the L. oceanica nuclear genome, indicating that mitochondrion-to-host-nucleus gene transfer occurred after the divergence of these two genera. Despite being of similar size and coding capacity, the level of synteny between L. oceanica and B. natans mtDNA is low, suggesting frequent rearrangements. Overall, our results suggest that chlorarachniophyte mtDNAs are more evolutionarily dynamic than their plastid counterparts.

  5. An Approach for Treating the Hepatobiliary Disease of Cystic Fibrosis by Somatic Gene Transfer

    Science.gov (United States)

    Yang, Yiping; Raper, Steven E.; Cohn, Jonathan A.; Engelhardt, John F.; Wilson, James M.

    1993-05-01

    Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.

  6. Lateral Gene Transfer in a Heavy Metal-Contaminated-Groundwater Microbial Community

    Science.gov (United States)

    Hemme, Christopher L.; Green, Stefan J.; Rishishwar, Lavanya; Prakash, Om; Pettenato, Angelica; Chakraborty, Romy; Deutschbauer, Adam M.; Van Nostrand, Joy D.; Wu, Liyou; He, Zhili; Jordan, I. King; Arkin, Adam P.; Kostka, Joel E.

    2016-01-01

    ABSTRACT Unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive. To delineate the importance of LGT in mediating the response of a groundwater microbial community to heavy metal contamination, representative Rhodanobacter reference genomes were sequenced and compared to shotgun metagenome sequences. 16S rRNA gene-based amplicon sequence analysis indicated that Rhodanobacter populations were highly abundant in contaminated wells with low pHs and high levels of nitrate and heavy metals but remained rare in the uncontaminated wells. Sequence comparisons revealed that multiple geochemically important genes, including genes encoding Fe2+/Pb2+ permeases, most denitrification enzymes, and cytochrome c553, were native to Rhodanobacter and not subjected to LGT. In contrast, the Rhodanobacter pangenome contained a recombinational hot spot in which numerous metal resistance genes were subjected to LGT and/or duplication. In particular, Co2+/Zn2+/Cd2+ efflux and mercuric resistance operon genes appeared to be highly mobile within Rhodanobacter populations. Evidence of multiple duplications of a mercuric resistance operon common to most Rhodanobacter strains was also observed. Collectively, our analyses indicated the importance of LGT during the evolution of groundwater microbial communities in response to heavy metal contamination, and a conceptual model was developed to display such adaptive evolutionary processes for explaining the extreme dominance of Rhodanobacter populations in the contaminated groundwater microbiome. PMID:27048805

  7. Gene transfer of the Na+,K+-ATPase β1 subunit using electroporation increases lung liquid clearance in rats

    OpenAIRE

    Machado-Aranda, David; Adir, Yochai; Young, Jennifer L.; Briva, A.; Budinger, G.R. Scott; Yeldandi, Anjana V.; Sznajder, Jacob I.; Dean, David A.

    2004-01-01

    The development of non-viral methods for efficient gene transfer to the lung is highly desired for the treatment of a number of pulmonary diseases. We have developed a non-invasive procedure using electroporation to transfer genes to the lungs of rats. Purified plasmid (100 to 600 μg) was delivered to the lungs of anesthetized rats through an endotracheal tube and a series of square wave pulses were delivered via electrodes placed on the chest. Relatively uniform gene expression was observed ...

  8. Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites

    OpenAIRE

    Pombert, Jean-François; Selman, Mohammed; Burki, Fabien; Bardell, Floyd T.; Farinelli, Laurent; Solter, Leellen F.; Whitman, Douglas W.; Weiss, Louis M.; Corradi, Nicolas; Patrick J Keeling

    2012-01-01

    Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to ex...

  9. Microbial co-habitation and lateral gene transfer: what transposases can tell us

    Energy Technology Data Exchange (ETDEWEB)

    Hooper, Sean D.; Mavromatis, Konstantinos; Kyrpides, Nikos C.

    2009-03-01

    Determining the habitat range for various microbes is not a simple, straightforward matter, as habitats interlace, microbes move between habitats, and microbial communities change over time. In this study, we explore an approach using the history of lateral gene transfer recorded in microbial genomes to begin to answer two key questions: where have you been and who have you been with? All currently sequenced microbial genomes were surveyed to identify pairs of taxa that share a transposase that is likely to have been acquired through lateral gene transfer. A microbial interaction network including almost 800 organisms was then derived from these connections. Although the majority of the connections are between closely related organisms with the same or overlapping habitat assignments, numerous examples were found of cross-habitat and cross-phylum connections. We present a large-scale study of the distributions of transposases across phylogeny and habitat, and find a significant correlation between habitat and transposase connections. We observed cases where phylogenetic boundaries are traversed, especially when organisms share habitats; this suggests that the potential exists for genetic material to move laterally between diverse groups via bridging connections. The results presented here also suggest that the complex dynamics of microbial ecology may be traceable in the microbial genomes.

  10. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

    Science.gov (United States)

    Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  11. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  12. Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

    Science.gov (United States)

    Mundell, Nathan A; Beier, Kevin T; Pan, Y Albert; Lapan, Sylvain W; Göz Aytürk, Didem; Berezovskii, Vladimir K; Wark, Abigail R; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T; Schier, Alexander F; Cepko, Constance L

    2015-08-01

    Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. PMID:25688551

  13. Progress in identifying a human ionizing-radiation repair gene using DNA-mediated gene transfer techniques

    International Nuclear Information System (INIS)

    The authors employing DNA-mediated gene transfer techniques in introducing human DNA into a DNA double-strand break (DSB) repair deficient Chinese hamster (CHO) cell mutant (xrs-6), which is hypersensitive to both X-rays (D0 = 0.39 Gy) and the antibiotic bleomycin (D0 = 0.01 μg/ml). High molecular weight DNA isolated from cultured human skin fibroblasts was partially digested with restriction enzyme Sau 3A to average sizes of 20 or 40 Kb, ligated with plasmid pSV2-gpt DNA, and transfected into xrs-6 cells. Colonies which developed under a bleomycin and MAX (mycophenolic acid/adenine/xanthine) double-selection procedure were isolated and further tested for X-ray sensitivity and DSB rejoining capacity. To date a total of six X-ray or bleomycin resistant transformants have been isolated. All express rejoining capacity for X-ray-induced DSB, similar to the rate observed for DSB repair in CHO wild type cells. DNA isolated from these primary transformants contain various copy numbers of pSV2-gpt DNA and also contain human DNA sequences as determined by Southern blot hybridization. Recently, a secondary transformant has been isolated using DNA from one of the primary transformants. Cellular and molecular characterization of this transformant is in progress. DNA from a genuine secondary transformant will be used in the construction of a DNA library to isolate human genomic DNA encoding this radiation repair gene

  14. High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut.

    Science.gov (United States)

    Hinnebusch, B Joseph; Rosso, Marie-Laure; Schwan, Tom G; Carniel, Elisabeth

    2002-10-01

    The acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non-sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co-infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high-frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10-3 after only 3 days of co-infection, and after 4 weeks 95% of co-infected fleas contained an average of 103 antibiotic-resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic-resistant Y. pestis strains recently isolated from plague patients in Madagascar. PMID:12406213

  15. Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

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    Rouy Zoé

    2011-11-01

    Full Text Available Abstract Background Legionella pneumophila is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of Legionella species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg of a single species, namely L. pneumophila Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1 and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis. Results We show that L. pneumophila Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between L. pneumophila strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different Legionella species. Conclusion Horizontal gene transfer among bacteria and from eukaryotes to L. pneumophila as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time.

  16. Improving access to intestinal stem cells as a step toward intestinal gene transfer.

    Science.gov (United States)

    Sandberg, J W; Lau, C; Jacomino, M; Finegold, M; Henning, S J

    1994-03-01

    In previous studies exploring the intestinal epithelium as a potential site for somatic gene therapy, we concluded that the mucus lining the intestine constitutes a significant barrier to any attempts at gene transfer via the lumenal route. The mucus problem is aggravated by the fact that the epithelial stem cells, which are the logical target for gene transfer, are located deep in the intestinal crypts. The goals of the current study were to develop procedures that would improve accessibility to the intestinal stem cells and which would effect in vivo mucus removal without damaging the underlying epithelium. Initial experiments involved evaluation of the use of distension to improve accessibility to the intestinal crypts and the use of the mucolytic agents dithiothreitol (DTT) and N-acetyl-cysteine (NAC) versus a control solution of phosphate-buffered saline (PBS) for mucus removal. Catheters were inserted in each end of 3-cm terminal ileal segments in anesthetized rats. Two milliliters of agent was instilled into the clamped segment for 2 min, removed, and repeated. Lumenal distension resulted in shortened villi with wider intervillus spacing, thereby improving crypt access. Both NAC and DTT washes removed significant mucus between the villi but failed to reach the crypt lumen. To enhance mucus release from the crypt lumen, pilocarpine was selected due to its cholinergic properties and preferential binding to muscarinic receptors on crypt goblet cells. Pilocarpine given intraperitoneally 30 min prior to the mucolytic or PBS wash resulted in significant eradication of mucus down into the crypt lumen. This effect was still evident 3-4 hr later provided the intestine remained undisturbed.

  17. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    Science.gov (United States)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  18. Improving access to intestinal stem cells as a step toward intestinal gene transfer.

    Science.gov (United States)

    Sandberg, J W; Lau, C; Jacomino, M; Finegold, M; Henning, S J

    1994-03-01

    In previous studies exploring the intestinal epithelium as a potential site for somatic gene therapy, we concluded that the mucus lining the intestine constitutes a significant barrier to any attempts at gene transfer via the lumenal route. The mucus problem is aggravated by the fact that the epithelial stem cells, which are the logical target for gene transfer, are located deep in the intestinal crypts. The goals of the current study were to develop procedures that would improve accessibility to the intestinal stem cells and which would effect in vivo mucus removal without damaging the underlying epithelium. Initial experiments involved evaluation of the use of distension to improve accessibility to the intestinal crypts and the use of the mucolytic agents dithiothreitol (DTT) and N-acetyl-cysteine (NAC) versus a control solution of phosphate-buffered saline (PBS) for mucus removal. Catheters were inserted in each end of 3-cm terminal ileal segments in anesthetized rats. Two milliliters of agent was instilled into the clamped segment for 2 min, removed, and repeated. Lumenal distension resulted in shortened villi with wider intervillus spacing, thereby improving crypt access. Both NAC and DTT washes removed significant mucus between the villi but failed to reach the crypt lumen. To enhance mucus release from the crypt lumen, pilocarpine was selected due to its cholinergic properties and preferential binding to muscarinic receptors on crypt goblet cells. Pilocarpine given intraperitoneally 30 min prior to the mucolytic or PBS wash resulted in significant eradication of mucus down into the crypt lumen. This effect was still evident 3-4 hr later provided the intestine remained undisturbed. PMID:8018747

  19. Multiple interkingdom horizontal gene transfers in Pyrenophora and closely related species and their contributions to phytopathogenic lifestyles.

    Directory of Open Access Journals (Sweden)

    Bao-Fa Sun

    Full Text Available Many studies have reported horizontal gene transfer (HGT events from eukaryotes, especially fungi. However, only a few investigations summarized multiple interkingdom HGTs involving important phytopathogenic species of Pyrenophora and few have investigated the genetic contributions of HGTs to fungi. We investigated HGT events in P. teres and P. tritici-repentis and discovered that both species harbored 14 HGT genes derived from bacteria and plants, including 12 HGT genes that occurred in both species. One gene coding a leucine-rich repeat protein was present in both species of Pyrenophora and it may have been transferred from a host plant. The transfer of genes from a host plant to pathogenic fungi has been reported rarely and we discovered the first evidence for this transfer in phytopathogenic Pyrenophora. Two HGTs in Pyrenophora underwent subsequent duplications. Some HGT genes had homologs in a few other fungi, indicating relatively ancient transfer events. Functional analyses indicated that half of the HGT genes encoded extracellular proteins and these may have facilitated the infection of plants by Pyrenophora via interference with plant defense-response and the degradation of plant cell walls. Some other HGT genes appeared to participate in carbohydrate metabolism. Together, these functions implied that HGTs may have led to highly efficient mechanisms of infection as well as the utilization of host carbohydrates. Evolutionary analyses indicated that HGT genes experienced amelioration, purifying selection, and accelerated evolution. These appeared to constitute adaptations to the background genome of the recipient. The discovery of multiple interkingdom HGTs in Pyrenophora, their significance to infection, and their adaptive evolution, provided valuable insights into the evolutionary significance of interkingdom HGTs from multiple donors.

  20. Parkinson-related parkin reduces α-Synuclein phosphorylation in a gene transfer model

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    Rebeck GW

    2010-11-01

    Full Text Available Abstract Background α-Synuclein aggregates in Lewy bodies and plays a central role in the pathogenesis of a group of neurodegenerative disorders, known as "Synucleinopathies", including Parkinson's disease. Parkin mutations result in loss of parkin E3-ubiquitin ligase activity and cause autosomal recessive early onset parkinsonism. Results We tested how these two genes interact by examining the effects of parkin on post-translational modification of α-Synuclein in gene transfer animal models, using a lentiviral gene delivery system into the striatum of 2-month old male Sprague Dawley rats. Viral expression of wild type α-Synuclein caused accumulation of α-Synuclein and was associated with increased cell death and inflammation. α-Synuclein increased PLK2 levels and GSK-3β activity and increased the levels of phosphorylated α-Synuclein and Tau. Parkin co-expression reduced the levels of phosphorylated α-Synuclein and attenuated cell death and inflammation. Parkin reduced PLK2 levels and increased PP2A activation. Conclusions These data suggest that parkin reduces α-Synuclein levels and alters the balance between phosphatase and kinase activities that affect the levels of phosphorylated α-Synuclein. These results indicate novel mechanisms for parkin protection against α-Synuclein-induced toxicity in PD.

  1. Adaptive horizontal transfer of a bacterial gene to an invasive insect pest of coffee.

    Science.gov (United States)

    Acuña, Ricardo; Padilla, Beatriz E; Flórez-Ramos, Claudia P; Rubio, José D; Herrera, Juan C; Benavides, Pablo; Lee, Sang-Jik; Yeats, Trevor H; Egan, Ashley N; Doyle, Jeffrey J; Rose, Jocelyn K C

    2012-03-13

    Horizontal gene transfer (HGT) involves the nonsexual transmission of genetic material across species boundaries. Although often detected in prokaryotes, examples of HGT involving animals are relatively rare, and any evolutionary advantage conferred to the recipient is typically obscure. We identified a gene (HhMAN1) from the coffee berry borer beetle, Hypothenemus hampei, a devastating pest of coffee, which shows clear evidence of HGT from bacteria. HhMAN1 encodes a mannanase, representing a class of glycosyl hydrolases that has not previously been reported in insects. Recombinant HhMAN1 protein hydrolyzes coffee berry galactomannan, the major storage polysaccharide in this species and the presumed food of H. hampei. HhMAN1 was found to be widespread in a broad biogeographic survey of H. hampei accessions, indicating that the HGT event occurred before radiation of the insect from West Africa to Asia and South America. However, the gene was not detected in the closely related species H. obscurus (the tropical nut borer or "false berry borer"), which does not colonize coffee beans. Thus, HGT of HhMAN1 from bacteria represents a likely adaptation to a specific ecological niche and may have been promoted by intensive agricultural practices. PMID:22371593

  2. Polygalacturonase from Sitophilus oryzae: Possible horizontal transfer of a pectinase gene from fungi to weevils

    Directory of Open Access Journals (Sweden)

    Zhicheng Shen

    2003-08-01

    Full Text Available Endo-polygalacturonase, one of the group of enzymes known collectively as pectinases, is widely distributed in bacteria, plants and fungi. The enzyme has also been found in several weevil species and a few other insects, such as aphids, but not in Drosophila melanogaster, Anopheles gambiae, or Caenorhabditis elegans or, as far as is known, in any more primitive animal species. What, then, is the genetic origin of the polygalacturonases in weevils? Since some weevil species harbor symbiotic microorganisms, it has been suggested, reasonably, that the symbionts' genomes of both aphids and weevils, rather than the insects' genomes, could encode polygalacturonase. We report here the cloning of a cDNA that encodes endo-polygalacturonase in the rice weevil, Sitophilus oryzae (L., and investigations based on the cloned cDNA. Our results, which include analysis of genes in antibiotic-treated rice weevils, indicate that the enzyme is, in fact, encoded by the insect genome. Given the apparent absence of the gene in much of the rest of the animal kingdom, it is therefore likely that the rice weevil polygalacturonase gene was incorporated into the weevil's genome by horizontal transfer, possibly from a fungus.

  3. Cholesteryl ester transfer protein levels and gene deficiency in Chinese patients with cardio-cerebrovascular diseases

    Institute of Scientific and Technical Information of China (English)

    庄一义; 汪俊军; 张宏娟; 李勇; 刘小传; 李露言; 陈光辉

    2002-01-01

    Objective To detect cholesteryl ester transfer protein (CETP) levels, frequencies of CETP D442G and Ⅰ14A mutations and characteristics of abnormal lipids in patients with cardio-cerebro vascular diseases. Methods Ninety-four myocardial infarction (MI) patients,110 stroke patients and 335 healthy controls were selected. The CETP concentration was determined using ELISA. The CETP activity was measured using a substrate of 14 C-radiolabeled discoidal bilayer particles. The CETP gene mutations were detected by PCR-RFLP. Results The CETP concentrations in the MI and stroke group, were higher than those in the controls. The gene mutation frequencies of D442G in the MI, stroke and control group were 3.5%, 3.6% and 5%, respectively, and the frequencies of Ⅰ14A were 1.05%, 0.91% and 1%, respectively. One case of D442G homozygote was detected in the healthy group. The frequency of two CETP gene mutations showed no significant difference among the patients and controls. The CETP concentration and activity in subjects with CETP mutations were one-third of those in the control group. The level of HDL-C, apo-A1 increased in the mutation subjects, while the TG level decreased. Conclusions The CETP level increased significantly in patients with cardio-cerebrovascular diseases. The carriers of CETP deficiency had CETP and lipid abnormalities.

  4. Improvement of Drought Tolerance in Transgenic Tobacco Plants by aDehydrin-Like Gene Transfer

    Institute of Scientific and Technical Information of China (English)

    SHENYe; JIAWei-long; ZHANGYan-qin; HUYuan-lei; WUQi; LINZhongping

    2004-01-01

    A full-length cDNA of dehydrin BcDh2 from Boea crassifolia and its antisense nucleotide sequence have been transferred into tobacco (Nicotiana tabacum) NC89 under the control of a caulifower mosaic virus 35S promoter. Under a progressive water stress, photosynthetic rate, transpiration rate and stomatal conductance of the sense and antisense plants reduced, and those of the control reduced much more. Photosynthetic rate, transpiration rate and stomatal conductance of all plants tested increased significantly 24 hours later after recoveried water supply, and those of the sense and antisense plants were higher than control. These indicated that overexpression of a dehydrin gene in tobacco may improve tolerance to water stress for plants, however, antisense BcDh2 gene in transgenic plant did not influence physiological conditions. The results of germination experiment of the transgenic seeds showed that on MS medium with different concentration PEG (8000), sense seed could more endure drought than control, while antisense seed was sensitive to drought. The results suggested that the overexpression of a dehydrin gene in tobacco might improve the tolerance to water stress for plants.

  5. Improvement of Drought Tolerance in Transgenic Tobacco Plants by a Dehydrin-Like Gene Transfer

    Institute of Scientific and Technical Information of China (English)

    SHEN Ye; JIA Wei-long; ZHANG Yan-qin; HU Yuan-lei; WU Qi; LIN Zhong-ping

    2004-01-01

    A full-length cDNA of dehydrin BcDh2 from Boea crassifolia and its antisense nucleotide sequence have been transferred into tobacco (Nicotiana tabacum) NC89 under the control of a caulifower mosaic virus 35S promoter. Under a progressive water stress, photosynthetic rate, transpiration rate and stomatal conductance of the sense and antisense plants reduced, and those of the control reduced much more. Photosynthetic rate, transpiration rate and stomatal conductance of all plants tested increased significantly 24 hours later after recoveried water supply, and those of the sense and antisense plants were higher than control. These indicated that overexpression of a dehydrin gene in tobacco may improve tolerance to water stress for plants, however, antisense BcDh2 gene in transgenic plant did not influence physiological conditions. The results of germination experiment of the transgenic seeds showed that on MS medium with different concentration PEG (8000), sense seed could more endure drought than control, while antisense seed was sensitive to drought. The results suggested that the overexpression of a dehydrin gene in tobacco might improve the tolerance to water stress for plants.

  6. Modeling horizontal gene transfer (HGT in the gut of the Chagas disease vector Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Durvasula Ravi V

    2011-05-01

    Full Text Available Abstract Background Paratransgenesis is an approach to reducing arthropod vector competence using genetically modified symbionts. When applied to control of Chagas disease, the symbiont bacterium Rhodococcus rhodnii, resident in the gut lumen of the triatomine vector Rhodnius prolixus (Hemiptera: Reduviidae, is transformed to export cecropin A, an insect immune peptide. Cecropin A is active against Trypanosoma cruzi, the causative agent of Chagas disease. While proof of concept has been achieved in laboratory studies, a rigorous and comprehensive risk assessment is required prior to consideration of field release. An important part of this assessment involves estimating probability of transgene horizontal transfer to environmental organisms (HGT. This article presents a two-part risk assessment methodology: a theoretical model predicting HGT in the gut of R. prolixus from the genetically transformed symbiont R. rhodnii to a closely related non-target bacterium, Gordona rubropertinctus, in the absence of selection pressure, and a series of laboratory trials designed to test the model. Results The model predicted an HGT frequency of less than 1.14 × 10-16 per 100,000 generations at the 99% certainty level. The model was iterated twenty times, with the mean of the ten highest outputs evaluated at the 99% certainty level. Laboratory trials indicated no horizontal gene transfer, supporting the conclusions of the model. Conclusions The model treats HGT as a composite event, the probability of which is determined by the joint probability of three independent events: gene transfer through the modalities of transformation, transduction, and conjugation. Genes are represented in matrices and Monte Carlo method and Markov chain analysis are used to simulate and evaluate environmental conditions. The model is intended as a risk assessment instrument and predicts HGT frequency of less than 1.14 × 10-16 per 100,000 generations. With laboratory studies that

  7. Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

    Science.gov (United States)

    Ou, Tong; Gao, Xiao-Chan; Li, San-Hua; Zhang, Qi-Ya

    2015-12-01

    The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L–170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host. PMID:26399243

  8. The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification.

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    Elisabet Vikeved

    2016-01-01

    Full Text Available The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT. Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania.To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species.LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets.

  9. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

    Directory of Open Access Journals (Sweden)

    Gideon Hen

    Full Text Available The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV, into the chorioallantoic membrane (CAM of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP or recombinant alpha-melanocyte-stimulating hormone (α-MSH genes, driven by the cytomegalovirus (CMV promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP nick end labeling (TUNEL assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA, and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

  10. Lateral transfer of a lectin-like antifreeze protein gene in fishes.

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    Laurie A Graham

    Full Text Available Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.

  11. New Markov Model Approaches to Deciphering Microbial Genome Function and Evolution: Comparative Genomics of Laterally Transferred Genes

    Energy Technology Data Exchange (ETDEWEB)

    Borodovsky, M.

    2013-04-11

    Algorithmic methods for gene prediction have been developed and successfully applied to many different prokaryotic genome sequences. As the set of genes in a particular genome is not homogeneous with respect to DNA sequence composition features, the GeneMark.hmm program utilizes two Markov models representing distinct classes of protein coding genes denoted "typical" and "atypical". Atypical genes are those whose DNA features deviate significantly from those classified as typical and they represent approximately 10% of any given genome. In addition to the inherent interest of more accurately predicting genes, the atypical status of these genes may also reflect their separate evolutionary ancestry from other genes in that genome. We hypothesize that atypical genes are largely comprised of those genes that have been relatively recently acquired through lateral gene transfer (LGT). If so, what fraction of atypical genes are such bona fide LGTs? We have made atypical gene predictions for all fully completed prokaryotic genomes; we have been able to compare these results to other "surrogate" methods of LGT prediction.

  12. Gene Loss and Horizontal Gene Transfer Contributed to the Genome Evolution of the Extreme Acidophile “Ferrovum”

    Science.gov (United States)

    Ullrich, Sophie R.; González, Carolina; Poehlein, Anja; Tischler, Judith S.; Daniel, Rolf; Schlömann, Michael; Holmes, David S.; Mühling, Martin

    2016-01-01

    Acid mine drainage (AMD), associated with active and abandoned mining sites, is a habitat for acidophilic microorganisms that gain energy from the oxidation of reduced sulfur compounds and ferrous iron and that thrive at pH below 4. Members of the recently proposed genus “Ferrovum” are the first acidophilic iron oxidizers to be described within the Betaproteobacteria. Although they have been detected as typical community members in AMD habitats worldwide, knowledge of their phylogenetic and metabolic diversity is scarce. Genomics approaches appear to be most promising in addressing this lacuna since isolation and cultivation of “Ferrovum” has proven to be extremely difficult and has so far only been successful for the designated type strain “Ferrovum myxofaciens” P3G. In this study, the genomes of two novel strains of “Ferrovum” (PN-J185 and Z-31) derived from water samples of a mine water treatment plant were sequenced. These genomes were compared with those of “Ferrovum” sp. JA12 that also originated from the mine water treatment plant, and of the type strain (P3G). Phylogenomic scrutiny suggests that the four strains represent three “Ferrovum” species that cluster in two groups (1 and 2). Comprehensive analysis of their predicted metabolic pathways revealed that these groups harbor characteristic metabolic profiles, notably with respect to motility, chemotaxis, nitrogen metabolism, biofilm formation and their potential strategies to cope with the acidic environment. For example, while the “F. myxofaciens” strains (group 1) appear to be motile and diazotrophic, the non-motile group 2 strains have the predicted potential to use a greater variety of fixed nitrogen sources. Furthermore, analysis of their genome synteny provides first insights into their genome evolution, suggesting that horizontal gene transfer and genome reduction in the group 2 strains by loss of genes encoding complete metabolic pathways or physiological features

  13. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    CERN Document Server

    Awazu, K; Tamiya, E

    2002-01-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of t