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Sample records for adenovirus-mediated gene transfer

  1. Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis

    E. Quattrocchi

    2011-09-01

    Full Text Available Collagen Induced Arthritis (CIA is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig, which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or β-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2×108 pfu suppressed established CIA, whereas the control β-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNg production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

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

    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.

  3. Adenovirus-mediated p53 gene transfer increases radiosensitivity of human gastric carcinoma cells

    Objective: To evaluate the effect of adenovirus-mediated p53 gene (Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines. Methods: Recombinant adenovirus carrying 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 and Western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. The four human gastric carcinoma cell line infected with Adp53 were irradiated with 4 Gy, and cell cycle distribution and apoptotic rate were assayed by flow cytometry. Nude mice xenograft models of W and M cell were intratumorally injected with Adp53 and 48 h later were irradiated with 6 Gy. Relative volume in growth curve of tumor was used to observe tumor regression. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection with Adp53 at 100 MOI, which caused high transfer rate of wild-type p53 and strong expression of P53 protein in the four human gastric carcinoma cell line cells. When evaluating radio-biologic efficacy by apoptotic rate, the apoptotic enhancement ratio of Adp53 at 4 Gy was 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell respectively, in vitro. The antitumor enhancement ratio of Adp53 at 6 Gy was 1.41 for cell-implanted tumor and 1.91 for M cell-implanted tumor in vivo. Conclusion: This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma

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

    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.

  5. Dual effects of adenovirus-mediated thrombopoietin gene transfer on hepatic oval cell proliferation and platelet counts

    Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P < 0.05) and reached its plateau at 9 days (P < 0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P < 0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells

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

    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.

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

    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.

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

    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.

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

    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

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

    Kim Samyong

    2005-05-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion The results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by

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

    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.

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

    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.

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

    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

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

    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.

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

    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)

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

    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.

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

    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...

  18. 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

    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

  19. Adenovirus-mediated p53 and ING4 gene co-transfer elicits synergistic antitumor effects through enhancement of p53 acetylation in breast cancer.

    Wu, Jie; Zhu, Yanbo; Xu, Chun; Xu, Hong; Zhou, Xiumin; Yang, Jicheng; Xie, Yufeng; Tao, Min

    2016-01-01

    Multigene-based combination therapy may be an effective practice in cancer gene therapy. Substantial studies have demonstrated that tumor suppressor p53 acetylation is indispensable for p53 activation. Inhibitor of growth 4 (ING4), as a novel tumor suppressor, is capable of remarkably enhancing p53 acetylation and its transcriptional activity. Hence, we assumed that combined treatment of p53 and ING4 double tumor suppressors would exhibit enhanced antitumor effects. The combined therapeutic efficacy of p53 and ING4 for human cancers has not been previously reported. We thus generated multiple promoter expression cassette-based recombinant adenovirus-co-expressing ING4 and p53 double tumor suppressor genes (AdVING4/p53), evaluated the combined effects of AdVING4/p53 on breast cancer using the MDA-MB-231 (mutant p53) human breast cancer cell line, and also elucidated its underlying molecular mechanisms. We demonstrated that AdVING4/p53-mediated p53 and ING4 co-expression induced synergistic growth inhibition and apoptosis as well as enhanced effects on upregulation of acetylated p53, P21, Bax, PUMA, Noxa, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, and downregulation of Bcl-2, CD31 and microvessel density (MVD) in MDA-MB-231 breast cancer in vitro and/or in vivo subcutaneous (s.c.) xenografted tumors. The synergistic antitumor activity elicited by AdVING4/p53 was closely associated with the enhanced activation of the intrinsic apoptotic pathway and synergistic inhibition of tumor angiogenesis, very possibly via ING4-mediated enhancement of p53 acetylation and activity. Thus, our results indicate that cancer gene therapy combining two or more tumor suppressors such as p53 and ING4 may constitute a novel and effective therapeutic modality for human breast cancer and other cancers. PMID:26530780

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

    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.

  1. Adenovirus-mediated IL-12 gene therapy in combination with radiotherapy for murine liver cancer

    Objective: To investigate the synergistic antitumor effects of adenovirus-mediated IL-12 gene therapy in combination with radiotherapy in mice bearing liver cancer. Methods: Balb/c mice bearing liver cancer received the treatment at day 1 with tumor local irradiation (TLI) of 20 Gy or mask irradiation when tumor size reached 0.6-1.0 cm. Within 1 hour after irradiation, adenovirus containing IL-12 gene or PBS was intra-tumor injected once a week. Forty-eight hours after the second injection, IFN-γ levels in sera and the supernatant of cultured spleen cells were assayed by ELISA, CTL activity of spleen cells was measured by 3H-TdR release assay, and phenotypes of tumor-infiltrating lymphocytes were analysed by immunohistochemical staining. Results: The growth of tumors in animals treated with a combination of IL-12 gene therapy and TLI was inhibited more significantly than those with either single treatment (P + and CD8+ lymphocyte infiltration and tumor-specific cytolytic activities, and the levels of IFN-γ in sera were higher in IL-12 gene therapy and IL-12 gene therapy combined with TLI groups. Conclusion: These results suggest that IL-12 gene therapy combined with radiotherapy is more effective than both single treatment modalities and can induce specific antitumor immuno-response greatly

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

    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.

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

    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.

  4. Combination of radiotherapy and adenovirus-mediated p53 gene therapy for MDM2-overexpressing hepatocellular carcinoma

    The p53 gene plays a determinant role in radiation-induced cell death and its protein product is negatively regulated by MDM2. We investigated whether adenovirus-mediated modified p53 gene transfer, which blocks p53-MDM2 binding, is effective for radiation-induced cell death in hepatocellular carcinoma (HCC) at different MDM2 cellular levels. Human hepatocellular carcinoma cell lines expressing MDM2 at low levels (Huh7) and high levels (SK-Hep1) were used. Ad-p53 and Ad-p53vp are replication-deficient adenoviral vectors containing human wild-type or modified p53, respectively. The anti-tumor effect was highest for Ad-p53 + radiotherapy (RT) in the low-level MDM2 cells, whereas this effect was highest for Ad-p53vp + RT in the MDM2-overexpressing cells. In Huh-7 cells, Ad-p53 + RT decreased cell viability (32%) in vitro and inhibited tumor growth (enhancement factor, 1.86) in vivo. Additionally, p21 expression and apoptosis were increased. In contrast, in SK-Hep1 cells, Ad-p53vp + RT showed decreased cell viability (51%) in vitro and inhibition of tumor growth (enhancement factor, 3.07) in vivo. Caspase-3 expression and apoptosis were also increased. Adenovirus-expressing modified p53, which blocks p53-MDM2 binding, was effective in killing tumor cells overexpressing MDM2. Furthermore, the combination strategy for disruption of the p53-MDM2 interaction with RT demonstrated enhanced anti-tumor effects both in vitro and in vivo. (author)

  5. Adenovirus-mediated gene transduction of truncated lκBα enhances radiosensitivity in human colon cancer cells

    Nuclear factor kappa B (NF-κB) is a transcription factor that is known to regulate apoptosis when cells are exposed to DNA-damaging agents such as ionizing radiation and cytotoxic drugs. We sought to determine if inhibition of NF-κB could enhance radiosensitivity in human colon cancer cells in vitro and in vivo. To inhibit NF-κB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IκBα (IκBαΔN) that lacks the phosphorylation sites essential for activation of NF-κB, and transfected two human colon cancer cell lines (HT29 and HCT15) with this vector. In vitro colony-forming assays revealed that the overexpression of the stable lκBα by AxIκBαΔN infection significantly suppressed cell growth after irradiation in both cell lines as compared to infection with a control vector, AxLacZ. Treatment with AxIκBαΔN and irradiation successfully inhibited the growth of HT29 xenografted subcutaneous tumors in nude mice with an 83.8% volume reduction on day 38 as compared to the untreated tumors. Furthermore, it was demonstrated that apoptosis was increased by adenovirus-mediated gene transduction of IκBαΔN in vitro and in vivo. These results indicated that inhibition of NF-κB could enhance radiosensitivity through an increase in radiation-induced apoptosis. We believe that radio-gene therapy using adenovirus-mediated gene transduction of IκBαΔN could be an attractive candidate as a treatment strategy for colorectal cancer. (author)

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

    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.

  7. 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

    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.

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

    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...

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

    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...

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

    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.

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

    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.

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

    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.

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

    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...

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

    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

  15. Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma

    Liu Ran-yi

    2012-12-01

    Full Text Available Abstract Background Interferon-γ (IFN-γ is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ, and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC cell lines in vitro and in xenografts model. Methods The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR, and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts. Results The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed. Conclusions These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma.

  16. Synergistic tumor suppression by adenovirus-mediated ING4/PTEN double gene therapy for gastric cancer.

    Zhang, H; Zhou, X; Xu, C; Yang, J; Xiang, J; Tao, M; Xie, Y

    2016-01-01

    Both inhibitor of growth 4 (ING4) and phosphatase and tensin homolog (PTEN) have been shown to be strong candidate tumor suppressors. However, the combined efficacy of ING4 and PTEN for human gastric cancer remains to be determined. In this report, we constructed a multiple promoter expression cassette-based recombinant adenovirus coexpressing ING4 and PTEN (AdVING4/PTEN), assessed the combined effects of AdVING4/PTEN on gastric cancer using wild-type p53 AGS and SNU-1 human gastric cancer cell lines, and elucidated its underlying mechanisms. We found that AdVING4/PTEN-induced synergistic growth inhibition and apoptosis in vitro AGS or SNU-1 tumor cells and in vivo AGS xenografted tumors subcutaneously inoculated in athymic BALB/c nude mice. Mechanistically, AdVING4/PTEN exhibited an enhanced effect on upregulation of p53, Ac-p53 (K382), P21, Bax, PUMA, Noxa, cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP as well as downregulation of Bcl-2 in vitro and in vivo. In addition, AdVING4/PTEN synergistically downregulated tumor vessel CD34 expression and reduced microvessel density, and additively inhibited vascular endothelial growth factor (VEGF) expression in vivo. The synergistic tumor suppression elicited by AdVING4/PTEN was closely associated with the synergistic induction of apoptosis possibly via enhancement of endogenous p53 responses through cooperatively facilitating p53's stability and acetylation, and the synergistic inhibition of tumor angiogenesis probably via overlapping reduction of VEGF through cooperatively downregulating hypoxia inducible factor-1α's level and transcription activity. Thus, our results indicate that cancer gene therapy combining ING4 and PTEN may constitute a novel and effective therapeutic modality for human gastric cancer and other cancers. PMID:26564429

  17. 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

    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

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

    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

  19. 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

    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

  20. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

    Ribault, S; Neuville, P; Méchine-Neuville, A; Augé, F; Parlakian, A; Gabbiani, G; Paulin, D; Calenda, V

    2001-03-16

    Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders. PMID:11249869

  1. Adenovirus-mediated transfer of p53 augments hyperthermia-induced apoptosis in U251 glioma cells

    Purpose: Hyperthermia kills glioma cells by inducing apoptosis and is thereby an effective therapeutic modality for the treatment of malignant gliomas. However, cells harboring mutated p53 are refractory to hyperthermia-induced apoptosis. In this study, we assessed whether or not adenovirus (Adv)-mediated transduction of p53 overrides this resistant mechanism. Methods and Materials: We transduced the p53 wild-type tumor suppressor gene into U251 glioma cells harboring mutated p53 using Adv vectors in combination with hyperthermia (43, 44.5 deg. C), and evaluated the degree of cell death and apoptosis. Results: The percentage of cells that had died, as measured by trypan blue staining, among U251 cells infected with the Adv for p53 (Adv-p53) and treated with hyperthermia, was significantly higher than the percentage of cells that had died among U251 cells infected with Adv-p53 and not treated with hyperthermia, or those infected with the control Adv for dE (Adv-dE) and treated with hyperthermia. The degree of apoptosis, measured at 24 h after treatment, in hyperthermia-treated U251 cells infected with Adv-p53 (43 deg. C, 73%; 44.5 deg. C, 92%) was much higher than that infected with Adv-p53 (41%), or that infected with control Adv-dE and treated with hyperthermia (43 deg. C, 1.3%; 44.5 deg. C, 19%). Treatment with combined hyperthermia and Adv-p53 infection induced cleavage of caspase-3 in U251 cells. Conclusion: These results indicate that Adv-mediated transduction of p53 would render glioma cells highly sensitive to hyperthermia

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

    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

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

    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.

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

    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 ...

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

    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...

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

    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)

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

    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

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

    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.

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

    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

  10. Overexpression of MicroRNA-30b Improves Adenovirus-Mediated p53 Cancer Gene Therapy for Laryngeal Carcinoma

    Liang Li

    2014-10-01

    Full Text Available MicroRNAs play important roles in laryngeal carcinoma and other cancers. However, the expression of microRNAs in paracancerous tissue has been studied less. Here, using laser capture microdissection (LCM, we detected the expression of microRNAs in paracancerous tissues. Among all down-regulated microRNAs in the center area of tumor tissues, only miR-30b expression was significantly reduced in paracancerous tissues compared to surgical margins. Therefore, to further investigate the effect of miR-30b on laryngeal carcinoma, we stably overexpressed miR-30b in laryngeal carcinoma cell line HEp-2 cells. It was found that although there was no significant difference in cell viability between miR-30b overexpressed cells and control HEp-2 cells, p53 expression was obviously enhanced in miR-30b overexpressed cells. Whether miR-30b could improve the anti-tumor effect of adenovirus-p53 (Ad-p53 in laryngeal carcinoma and other cancer cell lines was also evaluated. It was found that in miR-30b overexpressed HEp-2 cells, p53-mediated tumor cell apoptosis was obviously increased both in vitro and in vivo. MDM2-p53 interaction might be involved in miR-30b-mediated anti-tumor effect. Together, results suggested that miR-30b could modulate p53 pathway and enhance p53 gene therapy-induced apoptosis in laryngeal carcinoma, which could provide a novel microRNA target in tumor therapy.

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

    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...

  12. 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

    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

  13. 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

    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

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

    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.

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

    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.

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

    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

  17. The effects of combining ionizing radiation and adenovirus-mediated p53 gene transfer in human nasopharyngeal carcinoma cell lines

    Purpose/Objective: We have previously demonstrated that the introduction of human recombinant wild-type p53 carried by the adenoviral vector (Ad5CMV-p53) into two human nasopharyngeal carcinoma (NPC) cell lines (CNE-1 and CNE-2Z) resulted in significant cytotoxicity. In the current work, we wanted to evaluate the results of this strategy when combined with ionizing radiation (XRT). Materials and Methods: CNE-1, CNE-2Z, and a normal human nasopharyngeal fibroblast strain KS1, were infected with iso-effective doses of 2, 6 and 6 pfu/cell of Ad5CMV-p53 respectively. XRT was administered 24 hours post-infection, to coincide with the time of maximal recombinant p53 expression. Western blot analyses were conducted for p53, p21WAF1/CIP1, bax and bcl-2. Cell viability was evaluated using both the MTT and clonogenic assays. Presence of apoptosis was determined by using DNA agarose gel electrophoresis. Results: We observed that the combination of Ad5CMV-p53 + XRT (2, 4, and 6 Gy) resulted in an approximately 1-log greater level of cytotoxicity compared to that observed with XRT alone for both NPC cell lines. The MTT assay indicated sparing of the KS1 cells when subjected to the identical treatments. XRT alone stimulated minimal p53 expression; Ad5CMV-p53 alone induced significant recombinant p53 expression, which was not further enhanced by the addition of XRT. Similar observations were made for p21WAF1/CIP1 expression. No changes were observed for bax and bcl-2 expression with any of these treatments. Apoptosis was induced following 4 Gy of XRT alone, but was observed earlier, at 2 Gy when combined with Ad5CMV-p53. Conclusion: Additional cytotoxicity was observed for the NPC cell lines when XRT was combined with Ad5CMV-p53 infection, with concurrent sparing of normal cells (KS1). This cytotoxicity also appeared to be mediated through the induction of the apoptotic pathway. These results support our previous observation of the potential application of this strategy in the treatment of NPC, particularly when combined with XRT

  18. 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

    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

  19. 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

    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

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

    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.

  1. 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

    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

  2. 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

    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...

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

    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.

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

    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...

  5. 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

    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

  6. 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

    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

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

    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.

  8. 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

    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

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

    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.

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

    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.

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

    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...

  12. 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

    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

  13. Beta-Adrenergic gene therapy for cardiovascular disease

    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.

  14. Inferring horizontal gene transfer.

    Matt Ravenhall

    2015-05-01

    Full Text Available Horizontal or Lateral Gene Transfer (HGT or LGT is the transmission of portions of genomic DNA between organisms through a process decoupled from vertical inheritance. In the presence of HGT events, different fragments of the genome are the result of different evolutionary histories. This can therefore complicate the investigations of evolutionary relatedness of lineages and species. Also, as HGT can bring into genomes radically different genotypes from distant lineages, or even new genes bearing new functions, it is a major source of phenotypic innovation and a mechanism of niche adaptation. For example, of particular relevance to human health is the lateral transfer of antibiotic resistance and pathogenicity determinants, leading to the emergence of pathogenic lineages. Computational identification of HGT events relies upon the investigation of sequence composition or evolutionary history of genes. Sequence composition-based ("parametric" methods search for deviations from the genomic average, whereas evolutionary history-based ("phylogenetic" approaches identify genes whose evolutionary history significantly differs from that of the host species. The evaluation and benchmarking of HGT inference methods typically rely upon simulated genomes, for which the true history is known. On real data, different methods tend to infer different HGT events, and as a result it can be difficult to ascertain all but simple and clear-cut HGT events.

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

    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.

  16. Horizontal gene transfer in fungi

    Fitzpatrick, David A.

    2011-01-01

    Horizontal gene transfer (HGT) is frequently observed in prokaryotes and until recently was assumed to be of limited importance to eukaryotes. However, there is an increasing body of evidence to suggest that HGT is an important mechanism in eukaryotic genome evolution, particularly in unicellular organisms. The transfer of individual genes, gene clusters or entire chromosomes can have significant impacts on niche specification, disease emergence or shift in metabolic capabil...

  17. Does Gene Translocation Accelerate the Evolution of Laterally Transferred Genes?

    Hao, Weilong; Golding, G. Brian

    2009-01-01

    Lateral gene transfer (LGT) and gene rearrangement are essential for shaping bacterial genomes during evolution. Separate attention has been focused on understanding the process of lateral gene transfer and the process of gene translocation. However, little is known about how gene translocation affects laterally transferred genes. Here we have examined gene translocations and lateral gene transfers in closely related genome pairs. The results reveal that translocated genes undergo elevated ra...

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

    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

  19. Horizontal gene transfer in plants.

    Gao, Caihua; Ren, Xiaodong; Mason, Annaliese S; Liu, Honglei; Xiao, Meili; Li, Jiana; Fu, Donghui

    2014-03-01

    Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components. PMID:24132513

  20. 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

    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

  1. 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

    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

  2. Horizontal gene transfer in chromalveolates

    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.

  3. Adenovirus-mediated wild-type PTEN promoting glioma stem/progenitor cells autophagy activity

    ZHAO Yao-dong; Zi-long WEI; Zhang, Quan-Bin; LOU Mei-qing; HUANG, QIANG

    2013-01-01

    Background PTEN is an anti-oncogene frequently inactivating in glioma. The previous study found that PTEN was closely related to cellular autophagy activity. The purpose of this paper is to study whether the inactivation of PTEN in glioma stem/progenitor cells (GSPCs) is correlative with the low autophagic activity in GSPCs. Methods Wild-type PTEN genes were transferred into GSPCs mediated by adenovirus. The autophagic activity in GSPCs before or after the introduction of wild-type PTEN was...

  4. Efficient Gene Transfer in Bacterial Cell Chains

    Babic, Ana; Berkmen, Melanie B.; Lee, Catherine A.; Grossman, Alan D.

    2011-01-01

    Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathoge...

  5. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

  6. Transferring alien genes to wheat

    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

  7. Adenovirus-mediated wild-type PTEN promoting glioma stem/progenitor cells autophagy activity

    ZHAO Yao-dong

    2013-05-01

    Full Text Available Background PTEN is an anti-oncogene frequently inactivating in glioma. The previous study found that PTEN was closely related to cellular autophagy activity. The purpose of this paper is to study whether the inactivation of PTEN in glioma stem/progenitor cells (GSPCs is correlative with the low autophagic activity in GSPCs. Methods Wild-type PTEN genes were transferred into GSPCs mediated by adenovirus. The autophagic activity in GSPCs before or after the introduction of wild-type PTEN was detected by immunocytochemistry, electron microscopy, and Western blotting assay. Results After transfection of wild-type PTEN, a large number of microtuble-associated protein 1 light chain 3 (MAP1LC3-positive granules could be found in the cytoplasm of GSPCs under a confocal microscopy, and these granules were demonstrated to be autophagosomes under an electron microscope. Moreover, the expression of autophagy-related gene Beclin-1 significantly increased after the transfection of wild-type PTEN gene. Conclusion The inactivation of PTEN in GSPCs is one reason of the low autophagic activity of GSPCs.

  8. Translating Gene Transfer: A Stalled Effort

    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...

  9. Targeting Radiotherapy to Cancer by Gene Transfer

    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 ...

  10. Horizontal gene transfer between bacteria and animals

    Dunning Hotopp, Julie C.

    2011-01-01

    Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria to animal transfer occur as part of intimate relationships like those of endosymbionts and their invertebra...

  11. Horizontal gene transfer between bacteria and animals.

    Dunning Hotopp, Julie C

    2011-04-01

    Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria-to-animal transfer occur as part of intimate relationships such as those of endosymbionts and their invertebrate hosts, particularly insects and nematodes, while numerous transfers are also found in asexual animals. Both of these observations are consistent with modern evolutionary theory, in particular the serial endosymbiotic theory and Muller's ratchet. Although it is tempting to suggest that these particular lifestyles promote horizontal gene transfer, it is difficult to ascertain given the nonrandom sampling of animal genome sequencing projects and the lack of a systematic analysis of animal genomes for such transfers. PMID:21334091

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

    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

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

    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.

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

    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

  15. The ethics of human gene transfer.

    Kimmelman, Jonathan

    2008-03-01

    Almost 20 years since the first gene-transfer trial was carried out in humans, the field has made significant advances towards clinical application. Nevertheless, it continues to face numerous unresolved ethical challenges--among them are the question of when to initiate human testing, the acceptability of germline modification and whether the technique should be applied to the enhancement of traits. Although such issues have precedents in other medical contexts, they take on a different character in gene transfer, in part because of the scientific uncertainty and the social context of innovation. PMID:18278058

  16. Gene transfer in the GI tract and oral cavity

    Mullany, Peter

    2000-01-01

    Gene transfer is important in spreading antibiotic resistance and other traits such as virulence factors. In this review the molecular mechanisms of gene transfer are outlined and the biological consequences of bacterial gene transfer in the GI tract and the oral cavity (GIOC) are discussed. Finally areas of possible future research aimed at attaining a deeper understanding of the process of gene transfer and the potential for stopping or slowing unwanted transfer are discussed.Keywords: gene...

  17. Viral Vectors for in Vivo Gene Transfer

    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

  18. Rates of Lateral Gene Transfer in Prokaryotes

    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

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

    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.

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

    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.

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

    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.

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

    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.

  3. Horizontal gene transfer and bacterial diversity

    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.

  4. The use of alien gene transfers

    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)

  5. Gene Transfer with Poly-Melittin Peptides

    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...

  6. Gene transfer system for Rhodopseudomonas viridis.

    Lang, F S; Oesterhelt, D

    1989-01-01

    A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhiz...

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

    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.

  8. Evidences of lateral gene transfer between archaea and pathogenic bacteria

    Bokhari, Habib; Anwar, Maryam; Mirza, Hasan Bilal; Gillevet, Patrick Martin

    2011-01-01

    Acquisition of new genetic material through horizontal gene transfer has been shown to be an important feature in the evolution of many pathogenic bacteria. Changes in the genetic repertoire, occurring through gene acquisition and deletion, are the major events underlying the emergence and evolution of bacterial pathogens. However, horizontal gene transfer across the domains i.e. archaea and bacteria is not so common. In this context, we explore events of horizontal gene transfer between arch...

  9. DNA-mediated gene transfer without carrier DNA

    1981-01-01

    DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA- mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA- mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier- free trans...

  10. Progress in gene transfer by germ cells in mammals

    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.

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

    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...

  12. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside ...

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

    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

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

    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. Lentiviral vector gene transfer to porcine airways.

    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

  16. Optical gene transfer by femtosecond laser pulses

    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.

  17. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    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

  18. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    Daley, Daniel O; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first...

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

    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.

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

    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. Evolution of and Horizontal Gene Transfer in the Endornavirus Genus

    Song, Dami; Cho, Won Kyong; Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endor...

  2. Gene transfer approaches in cancer immunotherapy.

    Larin, S S; Georgiev, G P; Kiselev, S L

    2004-10-01

    The idea of enhancing or establishing effective immune response against endogenously developed tumor cells is not novel. More than a hundred years ago, bacterial components were used to develop antitumor immune response. Later, when a number of immune system-effecting cytokines had been discovered, they were used for systemic treatment of cancer patients. However, systemic treatment often resulted in even negative outcome. Recent developments of genetic approaches of cell modifications allowed developing of modern techniques of targeted tumor cell elimination. In the present paper, we review modern trends of the antitumor response enhancement based on immunoregulatory gene transfer into different cell types both in vivo and in vitro. Almost all these approaches are based on the activation of the adaptive arm of the immune system in response to tumor cells. However, recent studies indicate that the innate arm of the immune system, as well as adaptive arm, is involved in tumor suppression. The innate immune system uses nonrearranging germline receptors, which could trigger cellular effector responses that are conditional (or instructive) to the subsequent adaptive immune response. Last years' viewpoints on 'self' and 'non-self' recognition and primary induction of the immune response have changed. The key role of lymphocytes is pathogen recognition and, following immune response induction, switched on the central role of dendritic cells in 'non-self' recognition and induction of both innate and adaptive responses. Moreover, innate response is supposed to be an essential starting point in induction of successful and effective acquired response. Most cancer vaccines do not have 'non-self' marks presentation due to their endogenous origin, thus lacking their effectiveness in the induction of the specific long-lasting immune response. Taking this point into consideration, we can conclude that to make cancer vaccine more effective we have to present tumor antigens

  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

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

    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.

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

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

    2013-01-01

    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......, dramatically affecting the enzymes of core pathways, particularly amino acid and sugar metabolism, but also providing new genes of potential adaptive significance in the life of parasites. A broad range of prokaryotic donors is involved in such transfers, but there is clear and significant enrichment for...... bacterial groups that share the same habitats, including the human microbiota, as the parasites investigated. CONCLUSIONS: Our data show that ecology and lifestyle strongly influence gene origins and opportunities for gene transfer and reveal that, although the outlines of the core eukaryotic metabolism are...

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

    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

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

    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

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

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

    2015-01-01

    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 ...

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

    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.

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

    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. Electroporation-Mediated Gene Transfer Directly to the Swine Heart

    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...

  12. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    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...

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

    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

  14. Can we modify response to radiation therapy with gene transfer?

    Several recent studies suggest that gene transfer can be combined with irradiation to increase anti-tumor efficacy. Among genes of particular interest to be used in this combined approach are those involved in the regulation of radiation-induced lethality (apoptosis, DNA repair). Some additional aspects appear to be relatively specific to these combinations, such as the type of vector to be used (anaerobic bacteria) or the type of promoter (radio-inducible promoters). The first results obtained in mice bearing human xenograft tumors, combining gene transfer and irradiation are encouraging, but no clinical study has been performed so far. Finally it should be pointed out, in this area as well as in cancer gene therapy in general, that progress in gene vectorization is mandatory to optimize gene distribution within the tumor. (authors)

  15. Design of radiopharmaceuticals for monitoring gene transfer therapy

    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)

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

    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.

  17. 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

    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.

  18. Oncolytic adenovirus mediated Survivin knockdown by RNA interference suppresses human colorectal carcinoma growth in vitro and in vivo

    Wang Chun-Yi

    2009-06-01

    Full Text Available Abstract Background Colorectal cancer is a one of the most common alimentary malignancies. Survivin has been proved by many studies to be an ideal target for cancer gene therapy because of its strong anti-apoptotic effect. The reduction of Survivin expression by means of chemically synthesized small interfering RNA or small hairpin RNA expressed from plasmid and resulted growth inhibition of cancer cells had been proved by many studies including ours, but the transfection efficiency was not encouraging. So for the first time we constructed the Survivin shRNA into an oncolytic adenovirus, tested its effects on colorectal cancer cell lines and nude mice xenograft model. Methods In this study, we constructed an oncolytic adenovirus with a Survivin targeted small hairpin RNA and a reporter gene (ZD55-Sur-EGFP. The expression of Survivin mRNA and protein were analyzed by RT-PCR and western blot. The cell growth and apoptosis were tested by in vitro cytopathic assay, MTT assay and flow cytometry respectively. The effect of the constructed virus on xenograft model was evaluated by tumor volume and western blot analysis. Results ZD55-Sur-EGFP replicated in cancer cells specifically, reduced the expression of Survivin mRNA and protein expression effectively (P Conclusion We conclude Survivin RNA interference combining with oncolytic adenovirus virotherapy to be a promising treatment for colorectal cancer.

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

    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

  20. Gene transfer strategies for improving radiolabeled peptide imaging and therapy

    Rogers, B.E.; Buchsbaum, D.J. [Birmingham University of Alabama, Birmingham, AL (United States). Dept. of Radiation Oncology; Zinn, K.R. [Birmingham University of Alabama, Birmingham, AL (United States). Radiology

    2000-09-01

    Utilization of molecular biology techniques offers attractive options in nuclear medicine for improving cancer imaging and therapy with radiolabeled peptides. Two of these options include utilization of phage-panning to identify novel tumor specific peptides or single chain antibodies and gene transfer techniques to increase the antibodies and gene transfer techniques to increase the number of antigen/receptor sites expressed on malignant cells. The group has focused on the latter approach for improving radiolabeled peptide imaging and therapy. The most widely used gene transfer vectors in clinical gene therapy trials include retrovirus, cationic lipids and adenovirus. It has been utilized adenovirus vectors for gene transfer because of their ability to accomplish efficient in vivo gene transfer. Adenovirus vectors encoding the genes for a variety of antigens/receptors (carcinoembryonic antigen, gastrin-releasing peptide receptor, somatostatin receptor subtype 2 (SSTr2)) have all shown that their expression is increased on cancer cells both in vitro and in vivo following adenovirus infection. Of particular interest has been the adenovirus encoding for SSTr2 (AdCMVSSTr2). Various radioisotopes have been attached to somatostatin analogues for imaging and therapy of SSTr2-positive tumors both clinically and in animal models. The use of these analogues in combination with AdCMVSSTr2 is a promising approach for improving the detection sensitivity and therapeutic efficacy of these radiolabeled peptides against solid tumors. In addition, it has been proposed the use of SSTr2 as a marker for imaging the expression of another cancer therapeutic trans gene (e.g., cytosine deaminase, thymidine kinase) encoded within the same vector. This would allow for non-invasive monitoring of gene delivery to tumor sites.

  1. Agrobacterium-mediated gene transfer to Chrysanthemum.

    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

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

    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

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

    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.

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

    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

  5. Gene transfer strategies for improving radiolabeled peptide imaging and therapy

    Utilization of molecular biology techniques offers attractive options in nuclear medicine for improving cancer imaging and therapy with radiolabeled peptides. Two of these options include utilization of phage-panning to identify novel tumor specific peptides or single chain antibodies and gene transfer techniques to increase the antibodies and gene transfer techniques to increase the number of antigen/receptor sites expressed on malignant cells. The group has focused on the latter approach for improving radiolabeled peptide imaging and therapy. The most widely used gene transfer vectors in clinical gene therapy trials include retrovirus, cationic lipids and adenovirus. It has been utilized adenovirus vectors for gene transfer because of their ability to accomplish efficient in vivo gene transfer. Adenovirus vectors encoding the genes for a variety of antigens/receptors (carcinoembryonic antigen, gastrin-releasing peptide receptor, somatostatin receptor subtype 2 (SSTr2) have all shown that their expression is increased on cancer cells both in vitro and in vivo following adenovirus infection. Of particular interest has been the adenovirus encoding for SSTr2 (AdCMVSSTr2). Various radioisotopes have been attached to somatostatin analogues for imaging and therapy of SSTr2-positive tumors both clinically and in animal models. The use of these analogues in combination with AdCMVSSTr2 is a promising approach for improving the detection sensitivity and therapeutic efficacy of these radiolabeled peptides against solid tumors. In addition, it has been proposed the use of SSTr2 as a marker for imaging the expression of another cancer therapeutic transgene (e.g. cytosine deaminase, thymidine kinase) encoded within the same vector. This would allow for non-invasive monitoring of gene delivery to tumor sites

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

    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.

  7. Agrobacterium-mediated gene transfer to Chrysanthemum.

    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...

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

    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. Plastid evolution: gene transfer and the maintenance of 'stolen' organelles

    Archibald John M

    2010-06-01

    Full Text Available Abstract Many heterotrophic organisms sequester plastids from prey algae and temporarily utilize their photosynthetic capacity. A recent article in BMC Genomics reveals that the dinoflagellate Dinophysis acuminata has acquired photosynthesis-related genes by horizontal gene transfer, which might explain its ability to retain 'stolen' plastids for extended periods of time. See research article http://www.biomedcentral.com/1471-2164/11/366

  10. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    Getino Redondo, María; Sanabria Ríos, David J.; Fernández López, Raúl; Campos Gómez, Javier; Sánchez López, José M.; Fernández Medarde, Antonio; Carballeira Cabranes, Néstor M.; Cruz Calahorra, Fernando de la

    2015-01-01

    Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential fe...

  11. Plasmid-mediated horizontal gene transfer is a coevolutionary process

    Harrison, Ellie; Brockhurst, Michael A

    2012-01-01

    Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids ...

  12. Unconventional lateral gene transfer in extreme thermophilic bacteria

    César, Carolina Elvira; Bricio, Carlos; van Heerden, Esta; Littauer, Dereck; Berenguer, José; Álvarez, Laura

    2011-01-01

    Conjugation and natural competence are two major mechanisms that explain the acquisition of foreign genes throughout bacterial evolution. In recent decades, several studies in model organisms have revealed in great detail the steps involved in such processes. The findings support the idea that the major basis of these mechanisms is essentially similar in all bacteria. However, recent work has pinpointed the existence of new, evolutionarily different processes underlying lateral gene transfer....

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

    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

  14. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

    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.

  15. Horizontal gene transfer in the evolution of photosynthetic eukaryotes

    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.

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

    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

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

    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...

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

    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

  19. Examining Ancient Inter-domain Horizontal Gene Transfer

    Francisca C. Almeida

    2008-01-01

    Full Text Available Details of the genomic changes that occurred in the ancestors of Eukarya, Archaea and Bacteria are elusive. Ancient interdomain horizontal gene transfer (IDHGT amongst the ancestors of these three domains has been difficult to detect and analyze because of the extreme degree of divergence of genes in these three domains and because most evidence for such events are poorly supported. In addition, many researchers have suggested that the prevalence of IDHGT events early in the evolution of life would most likely obscure the patterns of divergence of major groups of organisms let alone allow the tracking of horizontal transfer at this level. In order to approach this problem, we mined the E. coli genome for genes with distinct paralogs. Using the 1,268 E. coli K-12 genes with 40% or higher similarity level to a paralog elsewhere in the E. coli genome we detected 95 genes found exclusively in Bacteria and Archaea and 86 genes found in Bacteria and Eukarya. These genes form the basis for our analysis of IDHGT. We also applied a newly developed statistical test (the node height test, to examine the robustness of these inferences and to corroborate the phylogenetically identifi ed cases of ancient IDHGT. Our results suggest that ancient inter domain HGT is restricted to special cases, mostly involving symbiosis in eukaryotes and specific adaptations in prokaryotes. Only three genes in the Bacteria + Eukarya class (Deoxyxylulose-5-phosphate synthase (DXPS, fructose 1,6-phosphate aldolase class II protein and glucosamine-6-phosphate deaminase and three genes–in the Bacteria + Archaea class (ABC-type FE3+ -siderophore transport system, ferrous iron transport protein B, and dipeptide transport protein showed evidence of ancient IDHGT. However, we conclude that robust estimates of IDHGT will be very difficult to obtain due to the methodological limitations and the extreme sequence saturation of the genes suspected of being involved in IDHGT.

  20. Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

    Cheng, Yilong; Wei, Hua; Tan, James-Kevin Y; Peeler, David J; Maris, Don O; Sellers, Drew L; Horner, Philip J; Pun, Suzie H

    2016-05-01

    The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery. PMID:27061622

  1. Can Viruses be Modified to Achieve Sustained Gene Transfer?

    HildegundCJErtl

    2011-07-01

    Full Text Available It is very easy to replace a faulty gene in an immunocompromised mouse. First, one takes a well-characterized virus, such as an adenovirus or an adeno-associated virus, and incorporates the correct version of the faulty gene together with some regulatory sequences into the genome. Then, one transduces the recombinant genome into helper cells, which will add the viral capsid. At last, one injects the resulting viral vector into the sick mouse, and the mouse is cured. It is not that easy in an immunocompetent mouse, let alone in a human, as over the eons the immune system evolved to eliminate viruses regardless if they penetrate as dangerous pathogens or are injected by a well-meaning gene therapist. Here we offer our perspective on the potential of how viral vectors achieve sustained gene transfer in the face of a hostile immune system.

  2. Characterization of an ancient lepidopteran lateral gene transfer.

    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.

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

    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.

  4. Gene Transfer To Intact Mesenteric Arteries by Electroporation

    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...

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

    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.

  6. Dynamic monitoring of horizontal gene transfer in soil

    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.

  7. Radiation improves gene transfer into human ovarian carcinoma cells

    Purpose/Objective: Poor gene transfer is the major stumbling block to successful gene therapy today. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. During studies to quantitate radiation activated recombination, we also found that both plasmid and adenoviral vector transduction could be increased by irradiation. The studies presented here describe the effects of irradiation on gene transduction efficiency (both transient and stable transduction) in several human ovarian carcinoma lines, as a prelude to in vivo animal studies. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human ovarian carcinoma cell lines (SKOV3, CAOV3 and PA1). Either irradiated or unirradiated cells were transfected with pRSVZ plasmid (containing a LacZ expression cassette) in either the supercoiled and linearized (XmnI) forms and β-galactosidase expression followed with time. Transfection efficiency was measured by flow cytometry following FDG staining at 0, 48, and 96 hours after irradiation. FDG is converted to a fluorescent metabolite by LacZ, and thus reflects the transfection efficiency of the LacZ containing vector. Vector quantitation was also performed by southern hybridization. Stable transduction efficiency was measured 14 -35 days after irradiation. Optimization of the time of irradiation with respect to transfection was performed. Since cells demonstrated increased stable recombination for as long as 96 hours after irradiation, continuous low dose rate and multiple radiation fractions were also tested. These experiments were repeated using the Ad5CMVlacZ. Dividing cells were exposed to Ad5CMVlacZ at an MOI of 0.1,1,5,10 and 100 to determine optimum transfection concentration. Transduction efficiency was again measured at various intervals to determine the radiation dose and interval post transfection which provides the maximum increase in transfection

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

    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

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

    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

  10. Gene Transfer into Mouse Prepancreatic Endoderm by Whole Embryo Electroporation

    Rousseau GG

    2005-03-01

    Full Text Available CONTEXT: Understanding gene function in the developing pancreas is a major issue for pancreatic cell therapy. The in vivo analysis of gene function has essentially been performed by modulating gene expression in transgenesis. A faster and easier method is electroporation of mouse embryos. This technique, coupled with whole embryo culture, enables one to deliver genes and analyze their effects in a spatially and temporally regulated manner. OBJECTIVE: We wanted to adapt the electroporation technique for gene transfer of whole e8.5 mouse embryos into the endoderm to allow expression of transgenes in the pancreas or liver. RESULTS: Using two platinum plate electrodes, low voltage and a precise positioning of the embryo in the electroporation cuvette we could target and express DNA constructs in the prepancreatic or prehepatic territories, identified with cell markers. We also demonstrated that this technique is a valuable tool in the study of transcriptional regulation in the developing endoderm. CONCLUSIONS: Targeted electroporation of whole embryos is a useful method of characterizing the gene network which controls pancreatic development.

  11. Monitoring of gene transfer for cancer therapy with radioactive isotopes

    Gene therapy for cancer has recently been developed, and four approaches are currently being evaluated in experimental and clinical studies: 1) protection of normal tissue, such as bone marrow, which are normally targets for cytotoxic drugs; 2) improvement of the host antitumor response by increasing the antitumor activity of tumor-infiltrating immuno-competent cells or by modifying the tumor cells to enhance their immunogenicity; 3) reversion of the malignant phenotype either by suppression of oncogene expression or by introduction of normal tumor suppressor genes; 4) direct killing of tumor cells by the transfer of cytotoxic or prodrug-activating genes. Monitoring of gene therapy by assessing metabolic effects or the uptake of a specific substance with radioactive isotopes is reviewed. The author's experience is mostly described: uptake measurements with 11 Cthymidine, 18FDG, 3-D-methylglucose, and methionine in the presence of different concentrations of ganciclovir after transfection of a rat hepatoma cell line with a retroviral vector containing the HSVtk gene. Non-suicide reporter gene approaches are also discussed. (K.H.)

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

    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

  13. Gene ontology based transfer learning for protein subcellular localization

    Zhou Shuigeng

    2011-02-01

    Full Text Available Abstract Background Prediction of protein subcellular localization generally involves many complex factors, and using only one or two aspects of data information may not tell the true story. For this reason, some recent predictive models are deliberately designed to integrate multiple heterogeneous data sources for exploiting multi-aspect protein feature information. Gene ontology, hereinafter referred to as GO, uses a controlled vocabulary to depict biological molecules or gene products in terms of biological process, molecular function and cellular component. With the rapid expansion of annotated protein sequences, gene ontology has become a general protein feature that can be used to construct predictive models in computational biology. Existing models generally either concatenated the GO terms into a flat binary vector or applied majority-vote based ensemble learning for protein subcellular localization, both of which can not estimate the individual discriminative abilities of the three aspects of gene ontology. Results In this paper, we propose a Gene Ontology Based Transfer Learning Model (GO-TLM for large-scale protein subcellular localization. The model transfers the signature-based homologous GO terms to the target proteins, and further constructs a reliable learning system to reduce the adverse affect of the potential false GO terms that are resulted from evolutionary divergence. We derive three GO kernels from the three aspects of gene ontology to measure the GO similarity of two proteins, and derive two other spectrum kernels to measure the similarity of two protein sequences. We use simple non-parametric cross validation to explicitly weigh the discriminative abilities of the five kernels, such that the time & space computational complexities are greatly reduced when compared to the complicated semi-definite programming and semi-indefinite linear programming. The five kernels are then linearly merged into one single kernel for

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

    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.

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

    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...

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

    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.

  17. Horizontal gene transfer of microbial cellulases into nematode genomes is associated with functional assimilation and gene turnover

    Dieterich Christoph

    2011-01-01

    Full Text Available Abstract Background Natural acquisition of novel genes from other organisms by horizontal or lateral gene transfer is well established for microorganisms. There is now growing evidence that horizontal gene transfer also plays important roles in the evolution of eukaryotes. Genome-sequencing and EST projects of plant and animal associated nematodes such as Brugia, Meloidogyne, Bursaphelenchus and Pristionchus indicate horizontal gene transfer as a key adaptation towards parasitism and pathogenicity. However, little is known about the functional activity and evolutionary longevity of genes acquired by horizontal gene transfer and the mechanisms favoring such processes. Results We examine the transfer of cellulase genes to the free-living and beetle-associated nematode Pristionchus pacificus, for which detailed phylogenetic knowledge is available, to address predictions by evolutionary theory for successful gene transfer. We used transcriptomics in seven Pristionchus species and three other related diplogastrid nematodes with a well-defined phylogenetic framework to study the evolution of ancestral cellulase genes acquired by horizontal gene transfer. We performed intra-species, inter-species and inter-genic analysis by comparing the transcriptomes of these ten species and tested for cellulase activity in each species. Species with cellulase genes in their transcriptome always exhibited cellulase activity indicating functional integration into the host's genome and biology. The phylogenetic profile of cellulase genes was congruent with the species phylogeny demonstrating gene longevity. Cellulase genes show notable turnover with elevated birth and death rates. Comparison by sequencing of three selected cellulase genes in 24 natural isolates of Pristionchus pacificus suggests these high evolutionary dynamics to be associated with copy number variations and positive selection. Conclusion We could demonstrate functional integration of acquired

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

    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.

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

    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.

  20. Study on magnetic gene transfer using HTS bulk magnet

    Highlights: •DNA–magnetite complexes were prepared as ferromagnetic DNA carrier. •The condition of magnetic field to suppress the diffusion was found by calculation. •The result of model experiment showed the validity of the calculated value. •The results of in vivo experiments showed that the amount of gene expression was significantly increased by magnetic field. -- Abstract: This study aimed to realize local and high-efficient gene expression by suppressing the diffusion of ferromagnetic DNA carriers in a strong magnetic field generated by HTS bulk magnet. DNA–magnetite complexes were prepared as ferromagnetic DNA carrier and the magnetic gene transfer using the DNA carriers was examined. From the results of the simulation and the model experiment, it was shown that the particle diffusion was suppressed within 10 mm in diameter by the magnetic field at 20 mm above the HTS bulk magnet. The results of in vivo experiments showed that the amount of gene expression was significantly increased by magnetic field

  1. Gene Transfer Enhancement by Alkylcarboxylation of Poly(propylenimine

    Maryam Hashemi

    2013-01-01

    Full Text Available Abstract Among synthetic carriers, dendrimers with the more flexible structure have attracted a great deal of researchers’ attention in the field of gene delivery. Followed by the promising results upon hydrophobic modification on polymeric structures in our laboratory, alkylcarboxylated poly (propylenimine-based carriers were synthesized by nucleophilic substitution of amines with alkyl moieties and were further characterized for their physicochemical and biological characteristics for plasmid DNA delivery. Although not noticeably effective gene transfer activity for hexanoate- and hexadecanoate-modified series was observed, but alkylation by decanoic acid significantly improved the transfection efficiency of the final constructs up to 60 fold in comparison with unmodified poly(propylenimine (PPI. PPI modified by 10-bromodecanoic acid at 50% grafting, showed significantly higher gene expression at c/p ratio of 2 compared to Superfect as positive control.  Overall, modification of PPI with 50% primary amines grafting with 10-bromodecanoic acid could increase the transfection efficiency which is occurred at lower c/p ratio when compared to Superfect, i.e. less amount of modified vector is required to exhibit the same efficiency as Superfect. Therefore, the obtained constructs seem to be safer carriers for long-term gene therapy applications.

  2. Association between translation efficiency and horizontal gene transfer within microbial communities

    Tuller, Tamir; Girshovich, Yana; Sella, Yael; Kreimer, Avi; Freilich, Shiri; Kupiec, Martin; Gophna, Uri; Ruppin, Eytan

    2011-01-01

    Horizontal gene transfer (HGT) is a major force in microbial evolution. Previous studies have suggested that a variety of factors, including restricted recombination and toxicity of foreign gene products, may act as barriers to the successful integration of horizontally transferred genes. This study identifies an additional central barrier to HGT—the lack of co-adaptation between the codon usage of the transferred gene and the tRNA pool of the recipient organism. Analyzing the genomic sequenc...

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

    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

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

    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

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

    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.

  6. Gene transfer into older chicken embryos by ex ovo electroporation.

    Luo, Jiankai; Yan, Xin; Lin, Juntang; Rolfs, Arndt

    2012-01-01

    The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos(1). Different structures such as DNA plasmids encoding genes(2-4), small interfering RNA (siRNA) plasmids(5), small synthetic RNA oligos(6), and morpholino antisense oligonucleotides(7) can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20--according to Hamburg and Hamilton)(8) and there are some disadvantages for its application in embryos at later stages (older than stage HH22--approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell. In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes(9) and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos(10-12). PMID:22872055

  7. Repeated, recent and diverse transfers of a mitochondrial gene to the nucleus in flowering plants.

    Adams, K L; Daley, D O; Qiu, Y L; Whelan, J; Palmer, J D

    2000-11-16

    A central component of the endosymbiotic theory for the bacterial origin of the mitochondrion is that many of its genes were transferred to the nucleus. Most of this transfer occurred early in mitochondrial evolution; functional transfer of mitochondrial genes has ceased in animals. Although mitochondrial gene transfer continues to occur in plants, no comprehensive study of the frequency and timing of transfers during plant evolution has been conducted. Here we report frequent loss (26 times) and transfer to the nucleus of the mitochondrial gene rps10 among 277 diverse angiosperms. Characterization of nuclear rps10 genes from 16 out of 26 loss lineages implies that many independent, RNA-mediated rps10 transfers occurred during recent angiosperm evolution; each of the genes may represent a separate functional gene transfer. Thus, rps10 has been transferred to the nucleus at a surprisingly high rate during angiosperm evolution. The structures of several nuclear rps10 genes reveal diverse mechanisms by which transferred genes become activated, including parasitism of pre-existing nuclear genes for mitochondrial or cytoplasmic proteins, and activation without gain of a mitochondrial targeting sequence. PMID:11099041

  8. Genetic modification of cereal crops by direct gene transfer

    On the basis of efficient in vitro culture and regeneration systems, reproducible transformation methods for different cereal crops were developed. Scutellar tissue of the immature embryos of hexaploid wheat and maize were used as targets for microprojectile mediated gene transfer. Bombardment of haploid microspores resulted in homozygous, transgenic and fertile barley plants. Each target was the subject of individual optimization processes of bombardment conditions by analysing the transient β-glucuronidase activity. Furthermore, phosphinothricin resistance conferred by the bar gene turned out to be a suitable selectable marker for regenerating transgenic crop plants. Summarizing the results of independent transformation experiments for wheat and maize led to a transformation efficiency of one transgenic plant per 83 and 230 bombardment immature embryos, respectively. For barley, the average of all the experiments was one transgenic plant per 2.8 x 1016 bombarded microspores. Primary transformants and progeny were analyzed for the enzyme activity of the two marker enzymes introduced and integration of the corresponding genes by Southern blot experiments. Stable integration of the foreign DNA and its inheritance by progeny were demonstrated. All the transformed plants showed normal morphology and their development and flowering were comparable with those of seed derived plants. (author). 28 refs, 2 tabs

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

    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 ...

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

    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

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

    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.

  12. Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles.

    Brevet, David; Hocine, Ouahiba; Delalande, Anthony; Raehm, Laurence; Charnay, Clarence; Midoux, Patrick; Durand, Jean-Olivier; Pichon, Chantal

    2014-08-25

    Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then L-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo. PMID:24853464

  13. Pathogen-origin horizontally transferred genes contribute to the evolution of Lepidopteran insects

    Li Zi-Wen; Shen Yi-Hong; Xiang Zhong-Huai; Zhang Ze

    2011-01-01

    Abstract Background Horizontal gene transfer (HGT), a source of genetic variation, is generally considered to facilitate hosts' adaptability to environments. However, convincing evidence supporting the significant contribution of the transferred genes to the evolution of metazoan recipients is rare. Results In this study, based on sequence data accumulated to date, we used a unified method consisting of similarity search and phylogenetic analysis to detect horizontally transferred genes (HTGs...

  14. Detecting rare gene transfer events in bacterial populations

    Kaare Magne Nielsen

    2014-01-01

    Full Text Available Horizontal gene transfer (HGT enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

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

    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. Genome-scale phylogenetic analysis finds extensive gene transfer among fungi

    Szöllősi, Gergely J.; Davín, Adrián Arellano; Tannier, Eric; Daubin, Vincent; Boussau, Bastien

    2015-01-01

    Although the role of lateral gene transfer is well recognized in the evolution of bacteria, it is generally assumed that it has had less influence among eukaryotes. To explore this hypothesis, we compare the dynamics of genome evolution in two groups of organisms: cyanobacteria and fungi. Ancestral genomes are inferred in both clades using two types of methods: first, Count, a gene tree unaware method that models gene duplications, gains and losses to explain the observed numbers of genes present in a genome; second, ALE, a more recent gene tree-aware method that reconciles gene trees with a species tree using a model of gene duplication, loss and transfer. We compare their merits and their ability to quantify the role of transfers, and assess the impact of taxonomic sampling on their inferences. We present what we believe is compelling evidence that gene transfer plays a significant role in the evolution of fungi. PMID:26323765

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

    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. Improved efficiency of the walnut somatic embryo gene transfer system.

    McGranahan, G H; Leslie, C A; Uratsu, S L; Dandekar, A M

    1990-01-01

    AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. PMID:24226275

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

    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

    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. Transduction-like gene transfer in the methanogen Methanococcus voltae

    Bertani, G.

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

  2. Heparin Inhibits Retrovirus Binding to Fibronectin as Well as Retrovirus Gene Transfer on Fibronectin Fragments

    Carstanjen, D.; Dutt, P; Moritz, T.

    2001-01-01

    Fibronectin fragments have been shown to improve retrovirus gene transfer efficiency by binding retrovirus and target cells. Using a novel virus adhesion assay, we confirmed binding of type C oncoretrovirus vectors to the heparin II domain of fibronectin and demonstrated inhibition of viral binding and gene transfer by heparin.

  3. Horizontal Gene Transfer and Its Part in the Reorganisation of Genetics during the LUCA Epoch

    Jheeta, Sohan

    2013-01-01

    Currently there are five known mechanisms of horizontal gene transfer (HGT): transduction, conjugation, transformation, gene transfer agents and membrane vesicle transfer. The question here is: what part did HGT play in the reorganisation of genetics during the last universal common ancestor (LUCA) epoch? LUCA is a construct to explain the origin of the three domains of life; namely Archaea, Bacteria and Eukarya. This editorial offers a general introduction to the relevance and ultimate signi...

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

    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.

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

    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.

  6. Proteasome Inhibitors Enhance Bacteriophage Lambda (λ) Mediated Gene Transfer in Mammalian Cells

    Volcy, Ketna; Dewhurst, Stephen

    2008-01-01

    Bacteriophage lambda vectors can transfer their genomes into mammalian cells, resulting in expression of phage-encoded genes. However, this process is inefficient. Experiments were therefore conducted to delineate the rate limiting step(s) involved, using a phage vector that contains a mammalian luciferase reporter gene cassette. The efficiency of phage-mediated gene transfer in mammalian cells was quantitated, in the presence or absence of pharmacologic inhibitors of cell uptake and degradat...

  7. On the need for widespread horizontal gene transfers under genome size constraint

    Stein Richard R

    2009-08-01

    Full Text Available Abstract Background While eukaryotes primarily evolve by duplication-divergence expansion (and reduction of their own gene repertoire with only rare horizontal gene transfers, prokaryotes appear to evolve under both gene duplications and widespread horizontal gene transfers over long evolutionary time scales. But, the evolutionary origin of this striking difference in the importance of horizontal gene transfers remains by and large a mystery. Hypothesis We propose that the abundance of horizontal gene transfers in free-living prokaryotes is a simple but necessary consequence of two opposite effects: i their apparent genome size constraint compared to typical eukaryote genomes and ii their underlying genome expansion dynamics through gene duplication-divergence evolution, as demonstrated by the presence of many tandem and block repeated genes. In principle, this combination of genome size constraint and underlying duplication expansion should lead to a coalescent-like process with extensive turnover of functional genes. This would, however, imply the unlikely, systematic reinvention of functions from discarded genes within independent phylogenetic lineages. Instead, we propose that the long-term evolutionary adaptation of free-living prokaryotes must have resulted in the emergence of efficient non-phylogenetic pathways to circumvent gene loss. Implications This need for widespread horizontal gene transfers due to genome size constraint implies, in particular, that prokaryotes must remain under strong selection pressure in order to maintain the long-term evolutionary adaptation of their "mutualized" gene pool, beyond the inevitable turnover of individual prokaryote species. By contrast, the absence of genome size constraint for typical eukaryotes has presumably relaxed their need for widespread horizontal gene transfers and strong selection pressure. Yet, the resulting loss of genetic functions, due to weak selection pressure and inefficient gene

  8. The Use of Viral Vectors in Gene Transfer Therapy

    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.

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

    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

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

    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

  11. Gene transfer from wild Helianthus to sunflower: topicalities and limits

    Breton Catherine

    2010-03-01

    Full Text Available Sunflower (2n=17 belongs to the Helianthus genus (Asteraceae. Wild Helianthus species display morphological variation for branching and stem number, for architecture and seed size, and for resistance to abiotic and biotic stresses due to which they thrive in different environments in North America. The genus is divided into botanical sections, two for annual as sunflower, and two for perennial species as Jerusalem artichoke that produces rhizomes (tubers. We explain the difficulties and successes obtained by crossing sunflower with these species to improve the agronomic traits of the sunflower crop. It is easier to cross the annual species than the perennials’ with sunflower. Several traits such as Cytoplasmic male sterility and restorer Rf-PET1 genes, Downy mildew resistance, Phomopsis resistance, Sclerotinia resistance, Rust resistance, and Orobanche resistance have already been introduced from annual species into sunflower crop, but the complex genomic organization of these species compared to sunflower limits their important potential. Perennial species are much more diverse, and their genomes display 2n, 4n, or 6n chromosomes for n 17. The realities of inter-specific hybridization are relatively disappointing due to the introgression lines that have low oil and low seed yield. We report here several attempts to introgress agronomic traits from these species to sunflower, and we present as a case study, an introgressed progenies from H. mollis, a diploid species with sessile small leaves. We constructed a preliminary genetic map with AFLP markers in 21 BC1 plants, and we then showed that some progenies display 6 to 44% of introgression from H. mollis. Although this study is promising due to the novel compact architecture of the progenies, we cannot estimate the transferability from H. mollis to other perennial Helianthus to improve sunflower.

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

    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.

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

    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.

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

    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.

  15. Can we modify response to radiation therapy with gene transfer?; Transfert de gene pour modifier la reponse a la radiotherapie

    Marangoni, E.; Bourhis, J. [Institut Gustave Roussy, Dept. de Radiotherapie, 94 - Villejuif (France); Bay, J.O.; Verrelle, P. [Centre Jean-Perrin, Lab. d' Oncologie Moleculaire, INSERM CRI 9502 EA 2145, 63 - Clermont-Ferrand (France)

    2000-06-01

    Several recent studies suggest that gene transfer can be combined with irradiation to increase anti-tumor efficacy. Among genes of particular interest to be used in this combined approach are those involved in the regulation of radiation-induced lethality (apoptosis, DNA repair). Some additional aspects appear to be relatively specific to these combinations, such as the type of vector to be used (anaerobic bacteria) or the type of promoter (radio-inducible promoters). The first results obtained in mice bearing human xenograft tumors, combining gene transfer and irradiation are encouraging, but no clinical study has been performed so far. Finally it should be pointed out, in this area as well as in cancer gene therapy in general, that progress in gene vectorization is mandatory to optimize gene distribution within the tumor. (authors)

  16. Rate of Gene Transfer From Mitochondria to Nucleus: Effects of Cytoplasmic Inheritance System and Intensity of Intracellular Competition

    Yamauchi, Atsushi

    2005-01-01

    Endosymbiotic theory states that mitochondria originated as bacterial intracellular symbionts, the size of the mitochondrial genome gradually reducing over a long period owing to, among other things, gene transfer from the mitochondria to the nucleus. Such gene transfer was observed in more genes in animals than in plants, implying a higher transfer rate of animals. The evolution of gene transfer may have been affected by an intensity of intracellular competition among organelle strains and t...

  17. Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

    Luo, Jiankai; Yan, Xin; Lin, Juntang; Rolfs, Arndt

    2012-01-01

    The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electrop...

  18. Transfer of engineered genes from crop to wild plants

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

    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 is a...

  19. Transfer of engineered genes from crop to wild plants

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

    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 is a...... sexual reproduction has been the basis for breeding almost all crops....

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

    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...

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

    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

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

    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.

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

    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.

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

    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.

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

    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

  6. Adenovirus-mediated delivery of bFGF small interfering RNA increases levels of connexin 43 in the glioma cell line, U251

    Liu Hongsheng

    2010-01-01

    Full Text Available Abstract Background bFGF is an important growth factor for glioma cell proliferation and invasion, while connexin 43 is implicated in the suppression of glioma growth. Correspondingly, gliomas have been shown to have reduced, or compromised, connexin 43 expression. Methods In this study, a bFGF-targeted siRNA was delivered to the glioma cell line, U251, using adenovirus (Ad-bFGF-siRNA and the expression of connexin 43 and its phosphorylation state were evaluated. U251 cells were infected with Ad-bFGF-siRNA (100, 50, or 25 MOI, and infection with adenovirus expressing green fluorescent protein (Ad-GFP at 100 MOI served as a control. Western blotting and immunofluorescence were used to detect the expression levels, phosphorylation, and localization of connexin 43 in U251 cells infected, and not infected, with Ad-bFGF-siRNA. Results Significantly higher levels of connexin 43 were detected in U251 cells infected with Ad-bFGF-siRNA at 100 and 50 MOI than in cells infected with Ad-GFP, and the same amount of connexin 43 was detected in Ad-GFP-infected and uninfected U251 cells. Connexin 43 phosphorylation did not differ between Ad-bFGF-siRNA-infected and uninfected U251 cells. However, the ratio of phosphorylated to unphosphorylated connexin 43 in Ad-bFGF-siRNA cells was lower, and connexin 43 was predominantly localized to the cytoplasm. Using a scrape loading dye transfer assay, more Lucifer Yellow was transferred to neighboring cells in the Ad-bFGF-siRNA treated group than in the control group. Conclusion To our knowledge, this is the first description of a role for connexin 43 in the inhibition of U251 growth using Ad-bFGF-siRNA.

  7. Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy

    Keyhani Nemat O; Song Jian; Bonner Carol A; Xie Gary; Jensen Roy A

    2004-01-01

    Abstract Background The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertica...

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

    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.)

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

    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...

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

    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...

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

    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...

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

    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.

  13. Retrovirus-Associated Heparan Sulfate Mediates Immobilization and Gene Transfer on Recombinant Fibronectin

    Lei, Pedro; Bajaj, Bharat; Andreadis, Stelios T.

    2002-01-01

    Recombinant retroviruses have been shown to bind to fibronectin (FN) and increase the efficiency of gene transfer to a variety of cell types. Despite recent work to optimize gene transfer on recombinant FN, the mechanism of retrovirus binding to FN and the interactions of target cells with the bound virus remain elusive. We investigated the roles of virus surface glycoprotein (gp70), cell-conditioned medium, and proteoglycans in mediating retrovirus binding to FN. We also examined the role of...

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

    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 ...

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

    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 ...

  16. Effect of Caenorhabditis elegans age and genotype on horizontal gene transfer in intestinal bacteria

    Portal-Celhay, Cynthia; Nehrke, Keith; Martin J. Blaser

    2013-01-01

    Horizontal gene transfer (HGT) between bacteria occurs in the intestinal tract of their animal hosts and facilitates both virulence and antibiotic resistance. A model in which both the pathogen and the host are genetically tractable facilitates developing insight into mechanistic processes enabling or restricting the transfer of antibiotic resistance genes. Here we develop an in vivo experimental system to study HGT in bacteria using Caenorhabditis elegans as a model host. Using a thermosensi...

  17. Plasmids as mediators of gene transfer in the genetic manipulation of gram-positive bacteria

    O'Hara, Seamus

    1989-01-01

    Lack of suitable gene transfer techniques hampers genetic improvement and analysis of several industrially and clinically important gram-positive bacteria. Techniques already developed are often difficult to reproduce and limited in application. This study examines the feasibility of expanding the techniques available through the use of conjugation as a broad host range gene transfer mechanism. Such systems have been developed for gram-negative bacteria. Theoretical and practical aspec...

  18. Radiosensitization of head/neck squamous cell carcinoma by adenovirus-mediated expression of dominant negative constructs of the Nbs1 protein

    Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. In order to test this hypothesis we have devised recombinant adenoviruses expressing various portions of the Nbs1 protein and assessed the ability of these viruses to increase the radiation sensitivity of HNSCC cells. Materials and Methods: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1(Nbs1-300, aa453 to aa754) into an adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line 011. These cells were evaluated for expression of the viral based constructs and assayed for growth rate and clonogenic survival following radiation exposure. Results: A constitutively expressed GFP gene in the viral backbone confirmed efficient uptake of the virus into the 011 cell line and Western blot confirmed the presence of the virally expressed Nbs1 and Nbs1-300. Following exposure to ionizing radiation cells infected with the Nbs1-300 virus showed a significant reduction in growth rate relative to cells infected with control virus. Surprisingly, this effect was even stronger with the full-length wild-type Nbs1 protein. Examination of clonogenic survival also demonstrated statistically significant sensitization, however the effects of the two constructs were distinct as Nbs1-300 expression resulted in reduction of the shoulder while expression of the full-length Nbs1 showed a change in the slope of the survival curve

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

    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

  20. Adenovirus-mediated FIR demonstrated TP53-independent cell-killing effect and enhanced antitumor activity of carbon-ion beams.

    Kano, M; Matsushita, K; Rahmutulla, B; Yamada, S; Shimada, H; Kubo, S; Hiwasa, T; Matsubara, H; Nomura, F

    2016-01-01

    Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late

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

    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.

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

    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.

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

    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.

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

    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

  5. Expression of transferred thymidine kinase genes is controlled by methylation.

    Christy, B.; Scangos, G

    1982-01-01

    Plasmid pTKx-1, containing the herpes simplex virus gene for thymidine kinase (TK) inserted into the BamHI site of plasmid pBR322, was introduced into Ltk- cells by calcium phosphate precipitation in the absence of carrier DNA. Line 101 is a TK+ derivative of Ltk- that contains multiple copies of pTKx-1 in a multimeric structure. A derivative of 101 that retained but no longer expressed the herpes simplex TK genes (termed 101BU1) and derivatives of line 101BU1 that reexpressed the genes (term...

  6. Evolutionary transfer of the chloroplast tufA gene to the nucleus.

    Baldauf, S L; Palmer, J D

    1990-03-15

    Evolutionary gene transfer is a basic corollary of the now widely accepted endosymbiotic theory, which proposes that mitochondria and chloroplasts originated from once free-living eubacteria. The small organellar chromosomes are remnants of larger bacterial genomes, with most endosymbiont genes having been either transferred to the nucleus soon after endosymbiosis or lost entirely, with some being functionally replaced by pre-existing nuclear genes. Several lines of evidence indicate that relocation of some organelle genes could have been more recent. These include the abundance of non-functional organelle sequences of recent origin in nuclear DNA, successful artificial transfer of functional organelle genes to the nucleus, and several examples of recently lost organelle genes, although none of these is known to have been replaced by a nuclear homologue that is clearly of organellar ancestry. We present gene sequence and molecular phylogenetic evidence for the transfer of the chloroplast tufA gene to the nucleus in the green algal ancestor of land plants. PMID:2314461

  7. Apical Gene Transfer into Quiescent Human and Canine Polarized Intestinal Epithelial Cells by Lentivirus Vectors

    Seppen, Jurgen; Barry, Simon C.; Klinkspoor, J. Henriette; Katen, Louis J.; Lee, Sum P; Garcia, J. Victor; Osborne, William R. A.

    2000-01-01

    Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

  8. The interconnection between biofilm formation and horizontal gene transfer

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

    2012-01-01

    . 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...... 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...

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

    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.

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

    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