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Sample records for adenoviral gene transfer

  1. Adenoviral Mediated Gene Transfer into the Dog Brain In Vivo

    Candolfi, Marianela; Kroeger, Kurt; Pluhar, Elizabeth; Liu, Chunyan; Barcia, Carlos; Bergeron, Josee; Puntel, Mariana; Curtin, James; McNiel, Elizabeth; Freese, Andrew; Ohlfest, John; Moore, Peter; Kuoy, William; Lowenstein, Pedro; Castro, Maria

    2007-01-01

    OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. D...

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

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

    2004-01-01

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

  3. Imaging expression of adenoviral HSV1-tk suicide gene transfer using the nucleoside analogue FIRU

    Substrates for monitoring HSV1-tk gene expression include uracil and acycloguanosine derivatives.The most commonly used uracil derivative to monitor HSV1-tk gene transfer is 1-(2-fluoro-2-deoxy-β-D-arabinofuranosyl)-5-[*I]iodouracil (fialuridine; I*-FIAU), where the asterisk denotes any of the radioactive iodine isotopes that can be used. We have previously studied other nucleosides with imaging properties as good as or better than FIAU, including 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-[*I]iodouracil (FIRU). The first aim of this study was to extend the biodistribution data of 123I-labelled FIRU. Secondly, we assessed the feasibility of detecting differences in HSV1-tk gene expression levels following adenoviral gene transfer in vivo with 123I-FIRU. 9L rat gliosarcoma cells were stably transfected with the HSV1-tk gene (9L-tk+). 123I-FIRU was prepared by radioiodination of 1-(2-fluoro-2-deoxy-β-D-ribofuranosyl)-5-tributylstannyl uracil (FTMRSU; precursor compound) and purified using an activated Sep-Pak column. Incubation of 9L-tk+ cells and the parental 9L cells with 123I-FIRU resulted in a 100-fold higher accumulation of radioactivity in the 9L-tk+ cells after an optimum incubation time of 4 h. NIH-bg-nu-xid mice were then inoculated subcutaneously with HSV1-tk (-) 9L cells or HSV1-tk (+) 9L-tk+ cells into both flanks. Biodistribution studies and gamma camera imaging were performed at 15 min and 1, 2, 4 and 24 h p.i. At 15 min, the tumour/muscle, tumour/blood and tumour/brain ratios were 5.2, 1.0 and 30.3 respectively. Rapid renal clearance of the tracer from the body resulted in increasing tumour/muscle, tumour/blood and tumour/brain ratios, reaching values of 32.2, 12.5 and 171.6 at 4 h p.i. A maximum specific activity of 22%ID/g tissue was reached in the 9L-tk+ tumours 4 h after 123I-FIRU injection. Two Ad5-based adenoviral vectors containing the HSV1-tk gene were constructed: a replication-incompetent vector with the transgene in the former E1 region

  4. Dual-expressing adenoviral vectors encoding the sodium iodide symporter for use in noninvasive radiological imaging of therapeutic gene transfer

    Introduction: Noninvasive analysis of therapeutic transgene expression is important for the development of clinical translational gene therapy strategies against cancer. To image p53 and MnSOD gene transfer noninvasively, we used radiologically detectable dual-expressing adenoviral vectors with the human sodium iodide symporter (hNIS) as the reporter gene. Methods: Dual-expressing adenoviral vectors were constructed with hNIS cloned into E3 region and therapeutic genes, either MnSOD or p53, recombined into the E1 region. Steady-state mRNA levels of hNIS were evaluated by real-time polymerase chain reaction. hNIS function was determined by iodide uptake assay and MnSOD, and p53 protein levels were assessed by Western blots. Results: 125I- accumulation resulting from hNIS expression in both Ad-p53-hNIS- and Ad-MnSOD-hNIS-infected MDA-MB-435 cells could be visualized clearly on phosphorimaging autoradiograph. Iodide accumulation increased with increasing adenovirus titer, and there was a linear correlation between iodide uptake and dose. p53 and MnSOD protein levels increased as a function of adenovirus titer, and there was a direct positive correlation between p53 and MnSOD expression and hNIS function. P53 and MnSOD overexpression inhibited cell growth in the dual-expressing adenoviral vector-infected cells. Conclusions: Radiological detection of hNIS derived from dual-expressing adenoviral vectors is a highly effective method to monitor therapeutic gene transfer and expression in a noninvasive manner

  5. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

    2003-01-01

    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer efficienc

  6. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle. PMID:12960972

  7. Vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers.

    Fishbein, Ilia; Forbes, Scott P; Adamo, Richard F; Chorny, Michael; Levy, Robert J; Alferiev, Ivan S

    2014-01-01

    In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of

  8. Adenoviral p53 gene transfer and gemcitabine in three patients with liver metastases due to advanced pancreatic carcinoma

    Thiede, Christian; Fischer, Rainer; Ehninger, Gerhard; Haag, Cornelie

    2007-01-01

    Background. Current therapies for adenocarcinoma of the pancreas do not improve the life expectancy of patients. Methods. In a non-randomized pilot trail we tested whether a local therapy based upon an adenoviral gene transfer of wild type p53 in combination with gemcitabine administration would be safe in patients with liver metastases due to pancreatic carcinoma. We report on the clinical course of three patients with respect to safety, tolerability and tumor response. Results. Transient grade III toxicities occurred with fever, leucopenia, elevation of AP, ALT, AST, GGT, while grade IV toxicity occurred for bilirubin only. Laboratory tests suggested disseminated intravascular coagulation in all three patients, but fine needle biopsies of liver did not show any histological evidence of thrombus or clot formation. Progression of liver metastases was documented in one and stable disease in another patient two months after treatment. However, a major improvement with regression of the indexed lesion by 80% occurred in a third patient after a single administration of 7.5×1012 viral particles, and time to progression was extended to six months. Conclusion. The combination therapy of viral gene transfer and chemotherapy temporarily controls and diminishes tumor burden. Improvement of the toxicity profile is necessary. Further trials are warranted to improve treatment and life expectancy of patients suffering from fatal diseases such as pancreatic carcinoma. PMID:18333108

  9. Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

    Fishbein, Ilia; Forbes, Scott P.; Adamo, Richard F.; Chorny, Michael; Levy, Robert J.; Alferiev, Ivan S.

    2014-01-01

    In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector r...

  10. Adenoviral gene delivery to primary human cutaneous cells and burn wounds

    Hirsch, Stephan Tobias Florian; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Marcus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinsträßer, Lars

    2006-01-01

    The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determine...

  11. Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice

    Champion, Hunter C.; Bivalacqua, Trinity J.; Greenberg, Stanley S.; Giles, Thomas D.; Hyman, Albert L.; Kadowitz, Philip J.

    2002-01-01

    It has been shown that mice deficient in the gene coding for endothelial nitric-oxide synthase (eNOS) have increased pulmonary arterial pressure and pulmonary vascular resistance. In the present study, the effect of transfer to the lung of an adenoviral vector encoding the eNOS gene (AdCMVeNOS) on pulmonary arterial pressure and pulmonary vascular resistance was investigated in eNOS-deficient mice. One day after intratracheal administration of AdCMVeNOS to eNOS−/− mice, there was an increase in eNOS protein, cGMP levels, and calcium-dependent conversion of l-arginine to l-citrulline in the lung. The increase in eNOS protein and activity in eNOS−/− mice was associated with a reduction in mean pulmonary arterial pressure and pulmonary vascular resistance when compared with values in eNOS-deficient mice treated with vehicle or a control adenoviral vector coding for β-galactosidase, AdCMVβgal. These data suggest that in vivo gene transfer of eNOS to the lung in eNOS−/− mice can increase eNOS staining, eNOS protein, calcium-dependent NOS activity, and cGMP levels and partially restore pulmonary arterial pressure and pulmonary vascular resistance to near levels measured in eNOS+/+ mice. Thus, the major finding in this study is that in vivo gene transfer of eNOS to the lung in large part corrects a genetic deficiency resulting from eNOS deletion and may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced. PMID:12237402

  12. Helper-dependent adenoviral vectors in experimental gene therapy*

    Józkowicz, Alicja; Dulak, Józef

    2005-01-01

    In the majority of potential applications gene therapy will require an effective transfer of a transgene in vivo resulting in high-level and long-term transgene expression, all in the absence of significant toxicity or inflammatory responses. The most efficient vehicles for delivery of foreign genes to the target tissues are modified adenoviruses. Adenoviral vectors of the first generation, despite the high infection efficacy, have an essential drawback: they induce strong immune response, wh...

  13. Adenoviral Vectors for Hemophilia Gene Therapy

    Brunetti-Pierri, N; Ng, Philip

    2013-01-01

    Hemophilia is an inherited blood clotting disorder resulting from deficiency of blood coagulation factors. Current standard of care for hemophilia patients is frequent intravenous infusions of the missing coagulation factor. Gene therapy for hemophilia involves the introduction of a normal copy of the deficient coagulation factor gene thereby potentially offering a definitive cure for the bleeding disorder. A variety of approaches have been pursued for hemophilia gene therapy and this review article focuses on those that use adenoviral vectors. PMID:24883229

  14. Treatment of colorectal and hepatocellular carcinomas by adenoviral mediated gene transfer of endostatin and angiostatin-like molecule in mice

    Schmitz, V; Wang, L.; Barajas, M. (Miguel); Gomar, C.; Prieto, J.; Qian, C

    2004-01-01

    Aim and method: In this study, we explored the responsiveness of different tumour entities (colorectal carcinoma (CRC), hepatocellular carcinoma (HCC), and the murine Lewis lung carcinoma (LLC)) to angiostatic antitumour treatment with two recombinant adenoviral vectors encoding angiostatin-like molecule (AdK1-3) and endostatin (Adendo).

  15. Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade.

    Romero, M I; Smith, G M

    1998-12-01

    This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function. PMID:10023440

  16. Adenoviral gene transfer corrects the ion transport defect in the sinus epithelia of a porcine CF model.

    Potash, Andrea E; Wallen, Tanner J; Karp, Philip H; Ernst, Sarah; Moninger, Thomas O; Gansemer, Nicholas D; Stoltz, David A; Zabner, Joseph; Chang, Eugene H

    2013-05-01

    Cystic fibrosis (CF) pigs spontaneously develop sinus and lung disease resembling human CF. The CF pig presents a unique opportunity to use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. In this study, we investigated the ion transport defect in the CF sinus and found that CF porcine sinus epithelia lack cyclic AMP (cAMP)-stimulated anion transport. We asked whether we could restore CF transmembrane conductance regulator gene (CFTR) current in the porcine CF sinus epithelia by gene transfer. We quantified CFTR transduction using an adenovirus expressing CFTR and green fluorescent protein (GFP). We found that as little as 7% of transduced cells restored 6% of CFTR current with 17-28% of transduced cells increasing CFTR current to 50% of non-CF levels. We also found that we could overcorrect cAMP-mediated current in non-CF epithelia. Our findings indicate that CF porcine sinus epithelia lack anion transport, and a relatively small number of cells expressing CFTR are required to rescue the ion transport phenotype. These studies support the use of the CF pig as a preclinical model for future gene therapy trials in CF sinusitis. PMID:23511247

  17. Effects of adenoviral gene transfer of mutated IкBα, a novel inhibitor of NF-кb, on human monocyte-derived dendritic cells

    Lin-fu ZHOU; Ming-shun ZHANG; Kai-sheng YIN; Yong JI; Wei-ping XIE; Xue-fan GUI; Xiao-hui JI

    2006-01-01

    Aim: To investigate the effects of adenoviral gene transfer of IкBα mutant (IкBαM), a novel inhibitor of nuclear factor кB (NF- кB), on apoptosis, phenotype and function of human monocyte-derived dendritic cells (DC). Methods: Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with Ad IкBαM or AdLacZ. The transcription and expression of the IкBαM gene, and the inhibitory effect of IкBαM on tumor necrosis factor (TNF)-α-induced NF-кB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction. Results: PCR and RT-PCR were used to detect a unique 801 bp band in Ad IкBαM-transfected mature DC, and also a dose- and time-dependent expression of the IкBαM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, Ad IкBαM significantly suppressed the TNF-α-induced NF- кB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC. Conclusion: Ad IкBαM effectively transfected and potently inhibited NF-кB activation in monocyte-derived mature DC. Overexpression of the IкBαM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.

  18. Prevention of beta cell dysfunction and apoptosis by adenoviral gene transfer of rat insulin-like growth factor 1

    CHEN Zhi-hong; LI Tang; CHEN Zong-bo; LUO Bing; SUN Ruo-peng

    2009-01-01

    Background Islet β-cells are almost completely destroyed when patients with type 1 diabete are diagnosed. To date, insulin substitute therapy is still one of the main treatments. The cure of type 1 diabetes requires β-cell regeneration from islet cell precursors and prevention of recurring autoimmunity, Therefore, β-cell regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes. Islet β-cell regeneration and development are controlled by many growth factors, especially insulin-like growth factor-1 (IGF-1).Methods Recombinant adenovirus encoding rat IGF-1 (rlGF-1) was constructed and transduced into rat β-cells, RINm5F cells. Western blotting analysis and ELISA were used to detect rlGF-1 protein. Streptozotocin (STZ) was used to induce RINm5F cell destruction. The level of nitric oxide (NO) was detected in cell culture supernatants by the Griess reaction. Islet cell function was evaluated by glucose-stimulated insulin production. Flow cytometry analysis was further used to investigate the apoptosis of RINm5F cells. Thiaoollyl blue viability assay was applied to determine cell viability.Results The recombined adenovirus-rlGF-1 was successfully constructed and the titer was 4.0×108pfu/ml. The rlGF-1 protein was effectively expressed in the RINm5F cells and cell culture supernatants, rlGF-1 expression remarkably inhibited STZ-induced islet cell apoptosis and significantly decreased the level of NO. Furthermore, IGF-1 expression also significantly protected insulin secretion and cell proliferation in a time-dependent manner.Conclusions Our study suggests that locally produced rlGF-1 from RINm5F cells may be beneficial in maintaining β-cell function, protecting β-cells from the destruction of apoptosis factors and promoting β-cell survival and proliferation. IGF-1 might be considered as a candidate gene in gene therapy for type 1 diabetes. In addition, it appears that the apoptosis induced by STZ may be NO-dependent.

  19. Adenoviral gene therapy in gastric cancer: A review

    Nima Khalighinejad; Hesammodin Hariri; Omid Behnamfar; Arash Yousefi; Amir Momeni

    2008-01-01

    Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promise to become the alternative choice of treatment in gastric cancer. In gene therapy, suicide genes, tumor suppressor genes and anti-angiogenesis genes among many others are introduced to cancer cells via vectors.Some of the vectors widely used in gene therapy are Adenoviral vectors. This review provides an update of the new developments in adenoviral cancer gene therapy including strategies for inducing apoptosis, inhibiting metastasis and targeting the cancer cells.

  20. Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

    Philip Ng

    2010-09-01

    Full Text Available Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application.

  1. Adenoviral Vectors for Hemophilia Gene Therapy

    Brunetti-Pierri, N.; Ng, Philip

    2013-01-01

    Hemophilia is an inherited blood clotting disorder resulting from deficiency of blood coagulation factors. Current standard of care for hemophilia patients is frequent intravenous infusions of the missing coagulation factor. Gene therapy for hemophilia involves the introduction of a normal copy of the deficient coagulation factor gene thereby potentially offering a definitive cure for the bleeding disorder. A variety of approaches have been pursued for hemophilia gene therapy and this review ...

  2. Clinical adenoviral gene therapy for prostate cancer

    Schenk, E.; Essand, M.; Bangma, Ch. H.; Barber, Ch.; Behr, J.-P.; Briggs, S.; Carlisle, R.; Cheng, W.-S.; Danielsson, A.; Dautzenberg, I. J. C.; Dzojic, H.; Erbacher, P.; Fisher, K.; Frazier, A.; Georgopoulos, L. J.; Hoeben, R.; Kochanek, S.; Koppers-Lalic, D.; Kraaij, R.; Kreppel, F.; Lindholm, L.; Magnusson, M.; Maitland, N.; Neuberg, P.; Nilsson, B.; Ogris, M.; Remy, J.-S.; Scaife, M.; Schooten, E.; Seymour, L.; Totterman, T.; Uil, T. G.; Ulbrich, Karel; Veldhoven-Zweistra, J. L. M.; de Vrij, J.; van Weerden, W.; Wagner, E.; Willemsen, R.

    2010-01-01

    Roč. 21, č. 7 (2010), s. 807-813. ISSN 1043-0342 EU Projects: European Commission(XE) 512087 - GIANT Keywords : adenovirus * gene delivery * prostate cancer Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.829, year: 2010

  3. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases. PMID:27245510

  4. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    Steinau Hans-Ulrich

    2011-01-01

    Full Text Available Abstract Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

  5. Tropism-modification strategies for targeted gene delivery using adenoviral vectors

    Baker, Andrew H; Parker, Alan L.; Bradshaw, Angela C.; Nicklin, Stuart A.; McNeish, Iain A; Raul Alba; Lynda Coughlan

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in...

  6. Helper-dependent adenoviral vectors for liver-directed gene therapy

    Brunetti-Pierri, Nicola; Ng, Philip

    2011-01-01

    Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory...

  7. Adenoviral-mediated localized CTLA-4Ig gene expression induces long-term allograft pancreas survival and donor-specific immune tolerance in rats

    2008-01-01

    T cell activation following alloantigen recognition plays a critical role in the development of the rejection in all solid organ, tissue and cell transplantation. A recombinant molecule, cytotoxic T lymphocyte antigen 4 antibody (CTLA-4Ig), is known to induce to T-cell into "anergy" by blocking the costimulatory B7-CD28 interaction. Either systemic or localized administration of CTLA-Ig has been shown to prolong allograft survival and induce donor-specific tolerance in some transplant models. In this study, we characterized the expression and immunosuppressive effectiveness of adenoviral-mediated CTLA-4Ig gene transfer. We demonstrated transduction of the allografts with AdCTLA-41g resulted in localized expression, permanent graft survival and stable donor-specific tolerance. In addition, by performing simultaneous dual-organ transplantation, we targeted on immunosuppression through a local expression of CTLA-4Ig via adenoviral-mediated gene transfer into pancreatic allografts.

  8. Challenges and Prospects for Helper-Dependent Adenoviral Vector-Mediated Gene Therapy

    Pasquale Piccolo; Nicola Brunetti-Pierri

    2014-01-01

    Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vect...

  9. Challenges and Prospects for Helper-Dependent Adenoviral Vector-Mediated Gene Therapy

    Pasquale Piccolo

    2014-04-01

    Full Text Available Helper-dependent adenoviral (HDAd vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.

  10. Adenoviral delivery of the EMX2 gene suppresses growth in human gastric cancer.

    Jie Li

    Full Text Available BACKGROUND: EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. RESULTS: In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. CONCLUSION: Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer.

  11. Adenoviral Delivery of the EMX2 Gene Suppresses Growth in Human Gastric Cancer

    Li, Jie; Mo, Minli; Chen, Zhao; Chen, Zhe; Sheng, Qing; Mu, Hang; Zhang, Fang; Zhang, Yi; Zhi, Xiu-Yi; Li, Hui; He, Biao; Zhou, Hai-Meng

    2012-01-01

    Background EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. Results In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. Conclusion Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer. PMID:23029345

  12. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  13. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie [Cancer Gene Therapy Group, Molecular Cancer Biology Program, Transplantation Laboratory, Haartman Institute, and Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki (Finland); Helsinki and Uusimaa Hospital District Laboratory, Helsinki University Central Hospital, Helsinki (Finland); Soliymani, Rabah [Protein Chemistry Unit, Department of Anatomy, Institute of Biomedicine, Biomedicum Helsinki (Finland); Tenhunen, Mikko [Department of Radiation and Oncology, Helsinki University Central Hospital, Helsinki (Finland); Ahtiainen, Laura [Cancer Gene Therapy Group, Molecular Cancer Biology Program, Transplantation Laboratory, Haartman Institute, and Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki (Finland); Helsinki and Uusimaa Hospital District Laboratory, Helsinki University Central Hospital, Helsinki (Finland); Hemminki, Akseli, E-mail: akseli.hemminki@helsinki.fi [Cancer Gene Therapy Group, Molecular Cancer Biology Program, Transplantation Laboratory, Haartman Institute, and Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki (Finland); Helsinki and Uusimaa Hospital District Laboratory, Helsinki University Central Hospital, Helsinki (Finland)

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  14. Adenoviral gene therapy in gastric cancer: A review

    Khalighinejad, Nima; Hariri, Hesammodin; Behnamfar, Omid; Yousefi, Arash; Momeni, Amir

    2008-01-01

    Gastric cancer is one of the most common malignancies worldwide. With current therapeutic approaches the prognosis of gastric cancer is very poor, as gastric cancer accounts for the second most common cause of death in cancer related deaths. Gastric cancer like almost all other cancers has a molecular genetic basis which relies on disruption in normal cellular regulatory mechanisms regarding cell growth, apoptosis and cell division. Thus novel therapeutic approaches such as gene therapy promi...

  15. Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy

    Siemionow Maria

    2006-08-01

    Full Text Available Abstract Background The aim of the present study was to develop and characterize a novel in vivo cancer gene therapy model in which intra-arterial adenoviral gene delivery can be characterized. In this model, the rat cremaster muscle serves as the site for tumor growth and provides convenient and isolated access to the tumor parenchyma with discrete control of arterial and venous access for delivery of agents. Results Utilizing adenovirus encoding the green fluorescent protein we demonstrated broad tumor transfection. We also observed a dose dependant increment in luciferase activity at the tumor site using an adenovirus encoding the luciferase reporter gene. Finally, we tested the intra-arterial adenovirus dwelling time required to achieve optimal tumor transfection and observed a minimum time of 30 minutes. Conclusion We conclude that adenovirus mediated tumor transfection grown in the cremaster muscle of athymic nude rats via an intra-arterial route could be achieved. This model allows definition of the variables that affect intra-arterial tumor transfection. This particular study suggests that allowing a defined intra-tumor dwelling time by controlling the blood flow of the affected organ during vector infusion can optimize intra-arterial adenoviral delivery.

  16. Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives

    Chen GX

    2014-10-01

    Full Text Available Guang-xia Chen,1,* Shu Zhang,2–4,* Xiao-hua He,1 Shi-yu Liu,1 Chao Ma,2–4 Xiao-Ping Zou2–4 1Department of Gastroenterology, First People’s Hospital of Xuzhou, Xuzhou, Jiangsu Province, People’s Republic of China; 2Department of Gastroenterology, Drum Tower Hospital, 3Medical School of Nanjing University, 4Jiangsu Clinical Medical Center of Digestive Disease, Nanjing, People’s Republic of China *These authors have contributed equally to the paperAbstract: Gene therapy has promised to be a highly effective antitumor treatment by introducing a tumor suppressor gene or the abrogation of an oncogene. Among the potential therapeutic transgenes, the tumor suppressor gene p53 serves as an attractive target. Restoration of wild-type p53 function in tumors can be achieved by introduction of an intact complementary deoxyribonucleic acid copy of the p53 gene using a suitable viral vector, in most cases an adenoviral vector (Adp53. Preclinical in vitro and in vivo studies have shown that Adp53 triggers a dramatic tumor regression response in various cancers. These viruses are engineered to lack certain early proteins and are thus replication defective, including Gendicine, SCH-58500, and Advexin. Several types of tumor-specific p53-expressing conditionally replicating adenovirus vectors (known as replication-competent CRAdp53 vectors have been developed, such as ONYX 015, AdDelta24-p53, SG600-p53, OBP-702, and H101. Various clinical trials have been conducted to investigate the safety and efficiency of these adenoviral vectors. In this review we will talk about the biological mechanisms, clinical utility, and therapeutic potentials of the replication-deficient Adp53-based and replication-competent CRAdp53-based gene therapy.Keywords: adenovirus, Adp53, CRAdp53

  17. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR Results in Severe Inflammation after Adenoviral Gene Therapy.

    Philipp Christian Seppelt

    Full Text Available Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs. In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1 in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR in order to reduce elastolysis.We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group. Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6 were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1 or β-galactosidase (Ad.β-Gal. As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI, and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM.IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43, but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00. Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001. As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001. However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated

  18. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

    Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

    2015-01-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  19. Comparison of osteogenic potentials of human rat BMP4 and BMP6 gene therapy using [E1-] and [E1-,E2b-] adenoviral vectors

    Hongwei Li, Jin Zhong Li, Debra D. Pittman, Andy Amalfitano, Gerald R. Hankins, Gregory A. Helm

    2006-01-01

    Full Text Available Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP first-generation adenoviral vectors (ADhBMPs are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6. A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6 and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6 adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.

  20. Effects of recombinant adenoviral vector containing IRE1α gene on proliferation and apoptosis of ATDC5 stem cells

    Xiang-zhu LI

    2013-09-01

    Full Text Available Objective To construct the recombinant adenoviral vector containing human IRE1α (type I transmembrane protein kinase/endoribonucleasegene, and investigate its effects on proliferation and apoptosis of ATDC5 stem cells. Methods  By using pAdEasyTM adenovirus vector system, the recombinant shuttle vectors of IRE1α full-length gene(pAdTrack-IRE1αand RNase+Kinasedomain(pAdTrack-R+Kwere constructed, and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmid pAd-IRE1α and pAd-R+K by electroporation method. Subsequently, the plasmids were transfected into HEK-293 cells to pack and amplify the recombinant adenovirus Ad-IRE1α and Ad-R+K. The expression of recombinant adenovirus was detected by PCR. The ATDC5 cells wereinfected in vitro by recombinant adenovirus Ad-IRE1α and Ad-R+K, the infection efficiency of green fluorescent protein(GFPwas observed, and the influence of Ad-IRE1α and Ad-R+K on the proliferation and apoptosis of ATDC5 cells under endoplasmic reticulum stress(ERS or non-ERS was detected by flow cytometry(FCM. Results Restriction endonuclease digestion analysis and PCR indicated that the recombinant adenovirus vector Ad-IRE1α andAd-R+K was successfully constructed. FCM detection showed that under ERS conditions, the G1 phasedcreased and S phase increased in ATDC5 cells after transfected by Ad-IRE1α and Ad-R+K, meanwhile the apoptosis rate increased significantly(P<0.05. Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5cells.

  1. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Andrew H. Baker

    2010-10-01

    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  2. Radiosensitization effect of recombinant adenoviral-mediated PUMA gene on pancreatic carcinoma cells

    Objective: To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods: The PANC-1 cells were infected with Ad- PUMA (MOI=10, 50 and 100, respectively) for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 cells were divided into 4 groups: control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups: control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotic index at different time points were recorded in 35 days. Results: The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant (MOI=10, mRNA=0.46± 0.02, protein=0.75± 0.09; MOI=50, mRNA=1.12±0.09, protein=1.01±0.18; MOI=100, mRNA=1.50±0.08, protein= 1.80±0.15; P3, (39.5±9.23)mm3, (33.6±10.3)mm3 and (52.0±11.43)mm3, respectively, P<0.05]. And the apoptotic index was increased in the same manner (AI=0.43±0.05, 0.29±0.10, 0.24±0.05 and 0.00±0.00, respectively, P<0.05). Conclusions: Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy. (authors)

  3. Generation and characterization of novel adenoviral vectors for hybrid nuclease-mediated gene targeting

    Henriques, Sara Filipa Dias

    2013-01-01

    Tese de mestrado [versão pública]. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2013 Adenovirus-based vectors are among the most efficient and disseminated gene transfer vehicles currently in use in a broad range of basic and applied research applications including the testing of gene therapy. As a gene therapy modality, gene targeting relies on the site-specific genome modification based on “integrases” or on the error-free homologous recombination...

  4. Comparison of [18F]FHBG and [14C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection

    Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells - stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [18F]FHBG, [3H]PCV, and [14C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [14C]FIAU accumulation was greater than that of [3H]PCV and [18F]FHBG in control cells and in HSV1-tk stably transfected cells (P8 pfu), [18F]FHBG and [3H]PCV accumulation was significantly greater than that of [14C]FIAU (P18F]FHBG and [3H]PCV accumulated to a significantly greater extent than [14C]FIAU in C6-stb-sr39tk+ and AdCMV-HSV1-sr39tk infected C6 cells (P14C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [18F]FHBG (P14C]FIAU, [18F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [18F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A-stb-tk+). [14C]FIAU accumulated in HSV1-tk stably expressing cells to a greater extent than either [3H]PCV or [18F]FHBG. However, the accumulation of [3H]PCV and [18F]FHBG in adenoviral infected C6 cells or hepatocytes was equivalent to or greater than that of [14C]FIAU. These results may be due to intracellular

  5. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  6. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  7. Adenoviral Producer Cells

    Imre Kovesdi

    2010-08-01

    Full Text Available Adenovirus (Ad vectors, in particular those of the serotype 5, are highly attractive for a wide range of gene therapy, vaccine and virotherapy applications (as discussed in further detail in this issue. Wild type Ad5 virus can replicate in numerous tissue types but to use Ad vectors for therapeutic purposes the viral genome requires modification. In particular, if the viral genome is modified in such a way that the viral life cycle is interfered with, a specific producer cell line is required to provide trans-complementation to overcome the modification and allow viral production. This can occur in two ways; use of a producer cell line that contains specific adenoviral sequences incorporated into the cell genome to trans-complement, or use of a producer cell line that naturally complements for the modified Ad vector genome. This review concentrates on producer cell lines that complement non-replicating adenoviral vectors, starting with the historical HEK293 cell line developed in 1977 for first generation Ad vectors. In addition the problem of replication-competent adenovirus (RCA contamination in viral preparations from HEK293 cells is addressed leading to the development of alternate cell lines. Furthermore novel cell lines for more complex Ad vectors and alternate serotype Ad vectors are discussed.

  8. 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.

  9. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

  10. Preparation of a recombinant adenoviral encoding human NIS gene and its specific expression in cardiomyocytes

    Objective: To construct a recombinant adenovirus vector containing the human NIS gene with the myosin light chain-2(MLC-2v) gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyocytes. Methods: MLC-2v promoter and NIS were subcloned into an adenovirus shuttle vector, and forwarded by homologous recombination in the bacteria BJ5183 containing AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the HEK293 cells to obtain recombinant adenovirus Ad-MLC-NIS. Ad-cytomegalovirus (CMV)-NIS with cytomegalovirus as the promoter, Ad-MLC without NIS and Ad-NIS without promoter were constructed as the controls. Cardiomyocytes and non-cardiomyocytes were then infected by the adenovirus. The protein expression was tested by Western blot analysis. The function and features of NIS protein were evaluated by dynamic iodide uptake and NaClO4 iodine uptake inhibition test in vitro. The viability and proliferation of cardiomyocytes after adenovirus transfection and radioiodine incubation were checked by trypan blue staining. Results: Recombinant NIS adenovirus was successfully constructed. Western blot analysis showed that the NIS protein was highly expressed in cardiomyocytes transfected with Ad-MLC-NIS, and all cells transfected with Ad-CMV-NIS. However, in non-cardiomyocytes transfected with Ad-MLC-NIS, little NIS protein was detected. Dynamic iodine uptake tests showed that the peaks of iodide uptake of the three different cell lines (H9C2, A549, U87 cell) transfected with Ad-MLC-NIS were 5844.0, 833.6 and 846.0 counts · min-1, respectively. The iodide uptake function of H9C2 was inhibited by NaClO4. There was almost no change in cell viability and proliferation when the MOI was 100. Conclusions: Ad-MLC-NIS allows myocardial specific expression of an external gene, and the cardiomyocytes with NIS expression are capable of iodine uptake. Further research of NIS as a reporter gene in

  11. Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

    Carr, David J; Wallace, Jacqueline M; Aitken, Raymond P; Milne, John S; Martin, John F; Zachary, Ian C; Peebles, Donald M; David, Anna L

    2016-06-01

    Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period. PMID:27103444

  12. Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson's Disease Model

    2006-01-01

    To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson's disease (PD), we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 (Ad-VEGF), and injected Ad-VEGF (or Ad-LacZ and PBS as controls) into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase (TH) immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals. It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.

  13. Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

    Dummer, R; Bergh, J; Karlsson, Y; Horovitz, JA; Mulder, NH; Huinin, DT; Burg, G; Hofbauer, G; Osanto, S

    2000-01-01

    p53 mutations are common genetic alterations in human cancer. Gene transfer of a wild-type (wt) p53 gene reverses the loss of normal p53 function in vitro and in vivo. A phase I dose escalation study of single intratumoral (i.t.) injection of a replication-defective adenoviral expression vector cont

  14. 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...

  15. Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli.

    Henriksen, Peter A; Hitt, Mary; Xing, Zhou; Wang, Jun; Haslett, Chris; Riemersma, Rudolph A; Webb, David J; Kotelevtsev, Yuri V; Sallenave, Jean-Michel

    2004-04-01

    Atherosclerosis is a chronic inflammatory disease affecting arterial vessels. Strategies to reduce the inflammatory responses of endothelial cells and macrophages may slow lesion development and prevent complications such as plaque rupture. The human protease human neutrophil elastase (HNE), oxidized low density lipoprotein, LPS, and TNF-alpha were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF-alpha and macrophage TNF-alpha production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-kappaB, through up-regulation of IkappaBalpha, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma. PMID:15034071

  16. INGN 201: Ad-p53, Ad5CMV-p53, adenoviral p53, p53 gene therapy--introgen, RPR/INGN 201.

    2007-01-01

    Introgen and its wholly owned European subsidiary Gendux AB are developing an adenoviral p53 gene therapy as a treatment for cancer in the US and Europe, respectively. Phase III trials in patients with head and neck cancer are ongoing, and a number of clinical trials in other cancer indications have been completed. INGN 201 is being reviewed by the EMEA for approval in Li-Fraumeni syndrome (LFS) under the provisions of exceptional circumstance; the therapy is available on a compassionate use basis to eligible LFS cancer patients under a protocol authorised by the US FDA. The p53 tumour suppressor gene is deleted or mutated in many tumour cells and is one of the most frequently mutated genes in human tumours. The p53 protein is one of the most intricate elements in the apoptotic signalling cascade, and a mutation in the gene encoding it is believed to result in a decreased ability of a cell to apoptose. Thus replacing this gene via adenovirally-mediated p53 gene therapy is hoped to result in increased apoptosis where it is administered.INGN 201 is available for licensing, although Introgen favours retaining partial or full rights to the therapy in the US. Introgen entered into a license agreement with The University of Texas System and MD Anderson Cancer Center in 1994. The technologies licenced include p53 and fus1 (INGN 401). The collaboration has yielded exclusive patent and licensing rights to numerous technologies. Introgen entered into a collaboration with Rhône-Poulenc Rorer Pharmaceuticals (now sanofi-aventis) to develop therapeutics based on p53 inhibition in October 1994. However, in June 2001 this relationship was restructured and Introgen assumed responsibility for the worldwide development of all p53 products including INGN 201, and acquired all marketing and commercialisation rights with respect to those products. Introgen initiated two phase III trials in head and neck cancer (in June 2000 and May 2001) at about 80 sites in the US, Canada and Europe

  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. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    Gahéry-Ségard, H; Molinier-Frenkel, V.; Le Boulaire, C; Saulnier, P.; Opolon, P; Lengagne, R.; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A.; Schatz, C; Courtney, M; Le Chevalier, T.; Tursz, T; Guillet, J G

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galact...

  19. 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.

  20. 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

  1. 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.

  2. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer.

    Binley, K; Iqball, S; Kingsman, A; Kingsman, S; Naylor, S

    1999-10-01

    Recombinant adenoviral vectors have a number of advantages for gene therapy, including transduction of a range of dividing and non-dividing cell types. However, this broad range may be a disadvantage if non-target cells are transduced and are adversely affected by expression of the transferred gene. Here we describe a novel adenoviral vector in which transcription of the transgene is restricted to the patho-physiological condition of low oxygen tension (hypoxia). Hypoxia activates the expression of a number of genes, principally via the stabilisation of members of the bHLH/PAS family of transcription factors that bind to a con- sensus DNA sequence, the hypoxia response element (HRE). We have configured an optimised HRE expression cassette into an adenoviral vector, AdOBHRE. A range of cell types, including primary human skeletal muscle, when transduced with AdOBHRE display a low basal level of transgene expression that is highly induced in hypoxia to levels equivalent to that obtained from the CMV promoter. The AdOBHRE vector could be exploited for transcriptionally targeted gene therapy for the treatment of diseases such as cancer, peripheral arterial disease, arthritis and anaemia where tissue hypoxia is a cardinal feature. PMID:10516721

  3. 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

  4. [Viral transfer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gene therapy].

    Wędrowska, Ewelina; Wandtke, Tomasz; Dyczek, Andrzej; Woźniak, Joanna

    2015-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces carcinoma cell death through the extrinsic pathway of apoptosis. Preclinical trials of gene therapy have been conducted using viral transfer of the TRAIL transgene into prostate, bladder, breast, kidney, liver, non-small cell lung cancer and also glioblastoma cells. Experiments in vitro demonstrated the extensive apoptosis of target cells as well as frequent disease regression or remission. TRAIL transfer did not show any side effects, opposite to chemotherapy. Encouraging results of TRAIL-related gene therapy were observed in rheumatoid arthritis and type 1 diabetes. Adenoviral vectors (AdV) encoding TRAIL are the most promising tool in anti-tumor therapy. They have undergone numerous modifications by increasing transfection efficiency and transgene expression in target cells. However, only one clinical phase I trial has been performed. AdV encoding the TRAIL transgene caused local inflammation and apoptosis in patients with prostate cancer. PMID:27259213

  5. 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.

  6. Expression of Mouse SCP2 Gene Adenoviral Vector Carrying Albumin Promoter in Hepa1-6 Cells%固醇携带蛋白2腺病毒载体的构建与鉴定

    贾岩峰; 崔云峰; 崔乃强; 彭雁飞; 宁召臣; 张琚

    2012-01-01

    Objectives To construct the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter, and study the relations between SCP2 gene and the formation of cholesterol calculus. Methods The cDNA of SCP2 gene was cloned by using RT-PCR technique. The albumin promoter was linked to SCP2 gene's upstream, and the EGFP gene lied in its downstream. The plasmid pDC312-ALB-SCP2-IRES2 -EGFP was constructed by the gene recombination technique. The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsCl method. The processes of TCID50 were applied to detect the titers of the adenoviral vectors. The RNA and protein were respectively extracted from the infected Hepal-6 cells by the adenoviral vector. The real-time quantitative PCR was employed to detect the mRNA expression levels, and the Western blotting analysis was used to measure the SCP2 protein levels. Result We constructed successfully the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter. When the mRNA levels of SCP2 gene were overexpressed, CYP7al mRNA levels were down-regulated (t=3.97,p<0.05); and the mRNA levels of HMGCR were up-regulated (t=3.23,p<0.05). Conclusions The SCP2 gene overexpression may affect cholesterol and bile acid metabolism, which could promote the formation of cholesterol calculus.%目的:构建携带白蛋白启动子SCP2 基因腺病毒载体,研究其与胆固醇结石形成的关系.方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2

  7. 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...

  8. Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

    Chiorini Jay A

    2010-01-01

    Full Text Available Abstract Background Pancreatic ductal adenocarcinoma (PDAC remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested. Methods We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines. Results The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6. Conclusion Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We

  9. Hematopoiesis stimulation test by interleukin 1α gene transfer in the Cynomolgus macaque: application to secondary medullary aplasia from an accidental irradiation

    After a description of the context of medullary aplasia (haematological radiobiology, radiation acute syndrome, therapeutic care), and an overview of knowledge about the interleukin-1 and medullary stroma cells, this research thesis aims at investigating therapeutic alternatives for radio-accidental aplasia. More precisely, it aims at defining means to get cytokines which are efficient for haematopoiesis. Interleukin-1 is chosen for its properties and tests are performed on a macaque with two approaches for gene transfer: an ex vivo transfer by retroviral vector enabling an integration in the target cell genome, and an in situ transfer by adeno-viral vector directly applied in the animal osseous medulla

  10. 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

  11. Viral vectors for vascular gene therapy

    Fischer, Lukas; Preis, Meir; Weisz, Anat; Koren, Belly; Lewis, Basil S; Flugelman, Moshe Y

    2002-01-01

    Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clini...

  12. Evaluation of the immune response to recombinant DNA vaccine and adenoviral vaccine co-expressing the M1 and HA genes of H5N1 influenza virus in mice.

    Guo, Jianqiang; Yao, Lihong; Chen, Aijun; Liu, Xiaoyu; Fu, Jinqi; Xu, Pengwei; Zhang, Zhiqing

    2011-06-01

    In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines. PMID:22034816

  13. 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...

  14. 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 ...

  15. Induction of specific humoral and cellular immune responses in a mouse model following gene fusion of HSP70C and Hantaan virus Gn and S0.7 in an adenoviral vector.

    Linfeng Cheng

    Full Text Available Heat shock proteins (HSPs display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV glycoprotein (GP and nucleocapsid protein (NP immunogenicity by heat shock protein 70 (HSP70, a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359-610 aa, HSP70C to the Gn and 0.7 kb fragment of the NP (aa1-274-S0.7. C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7 and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.

  16. 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...

  17. 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

  18. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral

  19. 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

  20. 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...

  1. 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

  2. Hematopoiesis stimulation test by interleukin 1{alpha} gene transfer in the Cynomolgus macaque: application to secondary medullary aplasia from an accidental irradiation; Essais de stimulation de l'hematopoiese par le transfert de gene de l'interleukine-1{alpha} chez le macaque cynomolgus: application a l'aplasie medullaire secondaire a une irradiation accidentelle

    De Revel, Th.

    2002-12-15

    After a description of the context of medullary aplasia (haematological radiobiology, radiation acute syndrome, therapeutic care), and an overview of knowledge about the interleukin-1 and medullary stroma cells, this research thesis aims at investigating therapeutic alternatives for radio-accidental aplasia. More precisely, it aims at defining means to get cytokines which are efficient for haematopoiesis. Interleukin-1 is chosen for its properties and tests are performed on a macaque with two approaches for gene transfer: an ex vivo transfer by retroviral vector enabling an integration in the target cell genome, and an in situ transfer by adeno-viral vector directly applied in the animal osseous medulla

  3. Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

    Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 'factory' cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products

  4. 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

  5. Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms.

    Helen N Jones

    Full Text Available Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor -1 (hIGF-1 in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined glucose transporter expression and localization in both a mouse model of IUGR and a model of human trophoblast, the BeWo Choriocarcinoma cell line.At gestational day 18, animals were divided into four groups; sham-operated controls, uterine artery branch ligation (UABL, UABL+Ad-hIGF-1 (10(8 PFU, UABL+Ad-LacZ (10(8 PFU. At gestational day 20, pups and placentas were harvested by C-section. For human studies, BeWo choriocarcinoma cells were grown in F12 complete medium +10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 hours. MOIs of 10∶1 and 100∶1 were utilized. The RNA, protein expression and localization of glucose transporters GLUT1, 3, 8, and 9 were analyzed by RT-PCR, Western blot and immunohistochemistry.In both the mouse placenta and BeWo, GLUT1 regulation was linked to altered protein localization. GLUT3, localized to the mouse fetal endothelial cells, was reduced in placental insufficiency but maintained with Ad-I GF-1 treatment. Interestingly, GLUT8 expression was reduced in the UABL placenta but up-regulated following Ad-IGF-1 in both mouse and human systems. GLUT9 expression in the mouse was increased by Ad-IGF-1 but this was not reflected in the BeWo, where Ad-IGF-1 caused moderate membrane relocalization.Enhanced GLUT isoform transporter expression and relocalization to the membrane may be an important mechanism in Ad-hIGF-1mediated correction of placental insufficiency.

  6. Sensitization of prostate cancer cell lines to 5-fluorocytosine induced by a replication incompetent adenoviral vector carrying a cytosine deaminase transcription unit

    2001-01-01

    AIM: To investigate the efficiency of cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines. METHODS: Cell culture, infectivity test and sensitivity test, observing the bystander effect and animal model experiment were carried out. RESULTS: All the established prostate cancer cell lines were eventually infectable, but ratio of vector/cell and time of exposed at which infection occurs was dependent on the cell lines. The expression of transfered cytosine deaminase gene peaked at different days, but persisted beyond 11 days. The prostate cell lines were sensitized to the 5-fluorocytosine by infection with the cytosine deaminase gene adenoviral vector, and only 5% of the LNCap and 10% of the RM-1 cells infected were required for 100% cell death. In the animal model, there was significant eradiation of tumor growth at the ratio of 400 vector particles/cell and with the systematic treatment of 5-fluorocytosine. CONCLUSION: The adenoviral vector carrying a cytosine deaminase transcription unit can sensitize the prostate cancer cell lines to 5-fluorocytosine, and the system can significantly inhibit the growth of prostatic tumor in mice.

  7. 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.

  8. 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)

  9. 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...

  10. Effects of AdCMV-p53 gene transfer induced by irradiation on cycle of human colorectal adenocarcinoma cells

    The work is to investigate effect of AdCMV-p53 gene transfer induced by 60Co γ-rays on cell cycles of human colorectal adenocarcinoma cells. HT-29 cells exposed to 0.5, 1.0 and 2.0 Gy were infected with AdCMV-GFP, a replication deficient recombinant adenoviral vector containing a CMV promoter and green fluorescent protein, or AdCMV-p53, a replication deficient recombinant adenoviral vector containing a CMV promoter and carrying human wild-type p53 gene. Survival rate of the cells was determined by clonogenic assay. Cell cycle and cell apoptosis were determined by flow cytometry. The results showed that, 0.5-1.0 Gy irradiation significantly enhanced the inhibition of AdCMV-p53 infection on HT-29. Compared with the control, 1 day after the infection, the cells in G0/G1 phase decreased by 5%-15%, the cells in S phase increased by 2%-19%. The 0.5 and 1.0 Gy irradiation made the cells in the in G2/M phase increase by 12%, infected with 80 MOI AdCMV-p5. There days later, the proportion of cells in G2/M phase in groups of 0.5 and 1.0 Gy irradiation +40 MOI AdCMV-p53 infection increased by 10%-13%. There was a relation between cell apoptosis and irradiation dose, or AdCMV-p53 dose. Therefore, the irradiation-induction could quicken the progression from G0/G1 phase to S phase, and promote S and G2/M phase arrest. (authors)

  11. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

    2010-01-01

    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  12. 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...

  13. 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...

  14. 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...

  15. The Evolution of Adenoviral Vectors through Genetic and Chemical Surface Modifications

    Cristian Capasso

    2014-02-01

    Full Text Available A long time has passed since the first clinical trial with adenoviral (Ad vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

  16. The evolution of adenoviral vectors through genetic and chemical surface modifications.

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-02-01

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges. PMID:24549268

  17. Enhanced Differentiation of Three-Gene-Reprogrammed Induced Pluripotent Stem Cells into Adipocytes via Adenoviral-Mediated PGC-1α Overexpression

    Yi-Jen Chen

    2011-11-01

    Full Text Available Induced pluripotent stem cells formed by the introduction of only three factors, Oct4/Sox2/Klf4 (3-gene iPSCs, may provide a safer option for stem cell-based therapy than iPSCs conventionally introduced with four-gene iPSCs. Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α plays an important role during brown fat development. However, the potential roles of PGC-1α in regulating mitochondrial biogenesis and the differentiation of iPSCs are still unclear. Here, we investigated the effects of adenovirus-mediated PGC-1α overexpression in 3-gene iPSCs. PGC-1α overexpression resulted in increased mitochondrial mass, reactive oxygen species production, and oxygen consumption. Microarray-based bioinformatics showed that the gene expression pattern of PGC-1α-overexpressing 3-gene iPSCs resembled the expression pattern observed in adipocytes. Furthermore, PGC-1α overexpression enhanced adipogenic differentiation and the expression of several brown fat markers, including uncoupling protein-1, cytochrome C, and nuclear respiratory factor-1, whereas it inhibited the expression of the white fat marker uncoupling protein-2. Furthermore, PGC-1α overexpression significantly suppressed osteogenic differentiation. These data demonstrate that PGC-1α directs the differentiation of 3-gene iPSCs into adipocyte-like cells with features of brown fat cells. This may provide a therapeutic strategy for the treatment of mitochondrial disorders and obesity.

  18. 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.

  19. Capsid modification strategies for detargeting adenoviral vectors.

    Parker, Alan L; Bradshaw, Angela C; Alba, Raul; Nicklin, Stuart A; Baker, Andrew H

    2014-01-01

    Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein. PMID:24132476

  20. 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...

  1. 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 ...

  2. Measurement of feline cytokines interleukin-12 and interferon- g produced by heat inducible gene therapy adenoviral vector using real time PCR

    Biologic tumor therapy using Interleukin-12 (IL-12) has shown promise as an adjuvant to radiation therapy. The goals for cancer gene immunotherapy include effective eradication of established tumors and generation of a lasting systemic immune response. Among the cytokines, IL-12 has been found to be most effective gene in eradicating experimental tumors, preventing the development of metastases, and eliciting long-term antitumor immunity. Depending on the tumor model, IL-12 can exert antitumor activities via T cells, NK cells or NKT cells. It induces the production of IFN-g and IFN-inducible protein-10. It is also postulated to have antiangiogenic effects, thus inhibiting tumor formation and metastases. However, its use in clinical trials has been restricted largely owing to its systemic hematologic and hepatotoxicity. We tested the efficacy of adenovirus mediated expression of feline IL-12 gene placed under the control of an inducible promoter, the heat shock proteins (hsp70B). This places gene expression under the control of an external physical agent (hyperthermia), thus offering an 'on-off' switch and potentially reducing systemic toxicity by restricting its expression locally to the tumor. Crandell Feline Kidney (CrFK) cells were infected using the construct and the supernatant was then used to stimulate production of interferon g (IFN-g) in feline peripheral blood mononuclear cells (PBMC). As there is no commercially available ELISA kit currently available to detect or measure feline cytokines, we used real time-PCR to measure cytokine mRNA. These results will be used to initiate a clinical trial in cats with soft tissue sarcomas examining hyperthermia Induced gene therapy in conjunction with radiation therapy. The real time- PCR techniques developed here will be used to quantitatively measure cytokine mRNA levels in the punch biopsy samples obtained from the cats during the clinical trial. Support for this study was in part by NCI grant CA72745

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35ΔRGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The αv-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-α, and interferon-α in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of αv-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes

  9. 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...

  10. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1±4.7%, 54.0±6.4%, 85.7±8.7%, and 98.4±1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704±6,659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168±2,134 picomole/106 cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression

  11. Comparison of human sodium iodide symporter (hNIS) gene expression between lentiviral and adenoviral vectors in rat mesenchymal stem cell

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been done. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated stably hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning the hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and Rad-hNIS transduced rMSC (adeno-hNIS-rMSC) was evaluated for the hNIS expression 48 hours post infection at MOI 1, 5, 20, 50, and 100. The hNIS expression in lenti-hNIS-rMSC or adeno-hNIS-rMSC was assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry using mono-clonal anti-hNIS antibody revealed that intensity of hNIS immunoreactivity in lenti-hNIS-rMSC was greater than that in adeno-hNIS-rMSC at MOl 20 but lower than that at MOl 50. Western blot analysis also showed that lenti-hNIS-rMSC was intermediate between adeno-hNIS-rMSCs at MOl 20 and 50 in hNIS expression. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (297046659 picomole/106 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (61682134 picomole/106 cells). These results suggest that lentivirus mediated hNIS expression is greater in terms of hNIS function but lower in terms of hNIS protein amount than adenovirus mediated hNIS expression 48 hours post infection. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative viral efficiency of transgene expression

  12. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Kim, Sung Jin; Lee, Heui Ran [Medical Research Center, Seoul National University, Seoul (Korea, Republic of)

    2008-10-15

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1{+-}4.7%, 54.0{+-}6.4%, 85.7{+-}8.7%, and 98.4{+-}1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704{+-}6,659 picomole/10{sup 6} cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168{+-}2,134 picomole/10{sup 6} cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

  13. 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.

  14. 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.

  15. Transduction of brain dopamine neurons by adenoviral vectors is modulated by CAR expression: rationale for tropism modified vectors in PD gene therapy.

    Travis B Lewis

    Full Text Available BACKGROUND: Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR. Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA neurons in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Ad5 was delivered to the substantia nigra (SN in wild type (wt and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the

  16. 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...

  17. Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

    Akbulut, Hakan; Zhang, Lixin; Tang, Yucheng; Deisseroth, Albert

    2003-05-01

    Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the

  18. 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

  19. 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...

  20. 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

  1. Immunocompromised Children with Severe Adenoviral Respiratory Infection

    Joanna C. Tylka

    2016-01-01

    Full Text Available Purpose. To investigate the impact of severe respiratory adenoviral infection on morbidity and case fatality in immunocompromised children. Methods. Combined retrospective-prospective cohort study of patients admitted to the intensive care unit (ICU in four children’s hospitals with severe adenoviral respiratory infection and an immunocompromised state between August 2009 and October 2013. We performed a secondary case control analysis, matching our cohort 1 : 1 by age and severity of illness score with immunocompetent patients also with severe respiratory adenoviral infection. Results. Nineteen immunocompromised patients were included in our analysis. Eleven patients (58% did not survive to hospital discharge. Case fatality was associated with cause of immunocompromised state (p=0.015, multiple organ dysfunction syndrome (p=0.001, requirement of renal replacement therapy (p=0.01, ICU admission severity of illness score (p=0.011, and treatment with cidofovir (p=0.005. Immunocompromised patients were more likely than matched controls to have multiple organ dysfunction syndrome (p=0.01, require renal replacement therapy (p=0.02, and not survive to hospital discharge (p=0.004. One year after infection, 43% of immunocompromised survivors required chronic mechanical ventilator support. Conclusions. There is substantial case fatality as well as short- and long-term morbidity associated with severe adenoviral respiratory infection in immunocompromised children.

  2. Generation of insulin-producing cells from the mouse liver using β cell-related gene transfer including Mafa and Mafb.

    Haruka Nagasaki

    Full Text Available Recent studies on the large Maf transcription factors have shown that Mafb and Mafa have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of Mafb before birth and of Mafa after birth or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an in vivo model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU mice. Adenoviral gene transfers of Pdx1/Neurod/Mafa (PDA and Pdx1/Neurod/Mafb (PDB combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that Mafa has a more sustainable role in insulin gene activation than does Mafb. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

  3. 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

  4. 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 ...

  5. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

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

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  6. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous {beta}-tricalcium phosphate ({beta}-TCP) material

    Uemura, Toshimasa; Kojima, Hiroko, E-mail: t.uemura@aist.go.jp [Nanosystem Research Institute (NRI), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Central-4, Tsukuba, Ibaraki 305-8562 (Japan)

    2011-06-15

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of {beta}-tricalcium phosphate ({beta}-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed {beta}-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous {beta}-TCP as a carrier.

  7. 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

  8. Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

    DelloRusso, Christiana; Scott, Jeannine M.; Hartigan-O'Connor, Dennis; Salvatori, Giovanni; Barjot, Catherine; Robinson, Ann S.; Robert W Crawford; Brooks, Susan V; Jeffrey S. Chamberlain

    2002-01-01

    Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin ex...

  9. 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...

  10. Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

    Gahéry-Ségard, H; Molinier-Frenkel, V; Le Boulaire, C; Saulnier, P; Opolon, P; Lengagne, R; Gautier, E; Le Cesne, A; Zitvogel, L; Venet, A; Schatz, C; Courtney, M; Le Chevalier, T; Tursz, T; Guillet, J G; Farace, F

    1997-01-01

    Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein. PMID:9410899

  11. Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation.

    Xing, Z; Braciak, T; Ohkawara, Y; Sallenave, J M; Foley, R; Sime, P J; Jordana, M; Graham, F L; Gauldie, J

    1996-04-01

    Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others. PMID:8613693

  12. 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.

  13. 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...

  14. 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

  15. 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)

  16. 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)

  17. 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.

  18. Selective homocysteine lowering gene transfer improves infarct healing, attenuates remodelling, and enhances diastolic function after myocardial infarction in mice.

    Ilayaraja Muthuramu

    Full Text Available BACKGROUND AND AIMS: Homocysteine levels predict heart failure incidence in prospective epidemiological studies and correlate with severity of heart failure in cross-sectional surveys. The objective of this study was to evaluate whether a selective homocysteine lowering intervention beneficially affects cardiac remodelling and cardiac function after myocardial infarction (MI in a murine model of combined hypercholesterolemia and hyperhomocysteinemia. METHODOLOGY AND PRINCIPAL FINDINGS: A selective homocysteine lowering gene transfer strategy was evaluated in female C57BL/6 low density lipoprotein receptor (Ldlr⁻/⁻ cystathionine-ß-synthase (Cbs⁺/⁻ deficient mice fed a hyperhomocysteinemic and high saturated fat/high cholesterol diet using an E1E3E4-deleted hepatocyte-specific adenoviral vector expressing Cbs (AdCBS. MI was induced by permanent ligation of the left anterior descending coronary artery 14 days after saline injection or gene transfer. AdCBS gene transfer resulted in a persistent more than 5-fold (p<0.01 decrease of plasma homocysteine levels and significantly improved endothelial progenitor cell function. Selective homocysteine lowering enhanced infarct healing as indicated by a 21% (p<0.01 reduction of infarct length at day 28 after MI and by an increased number of capillaries and increased collagen content in the infarct zone. Adverse remodelling was attenuated in AdCBS MI mice as evidenced by a 29% (p<0.05 reduction of left ventricular cavity area at day 28, by an increased capillary density in the remote myocardium, and by reduced interstitial collagen. The peak rate of isovolumetric relaxation was increased by 19% (p<0.05 and the time constant of left ventricular relaxation was reduced by 21% (p<0.05 in AdCBS MI mice compared to control MI mice, indicating improved diastolic function. CONCLUSION/SIGNIFICANCE: Selective homocysteine lowering gene transfer improves infarct healing, attenuates remodelling, and

  19. 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.

  20. GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

    Honda, Tsuyoshi; Coppola, Simona; Ghibelli, Lina; Cho, Song H; Kagawa, Shunsuke; Spurgers, Kevin B; Brisbay, Shawn M; Roth, Jack A; Meyn, Raymond E; Fang, Bingliang; McDonnell, Timothy J

    2004-04-01

    The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. PMID:15002033

  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. Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

    Benet, Marta; Jover, Ramiro; Bort, Roque

    2015-01-01

    Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses. PMID:26272145

  3. 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

  4. 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

  5. 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.

  6. 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...

  7. 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.

  8. Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

    Xiao-Bo Chen; Yong-Xiang Li; Yang Jiao; Wei-Ping Dong; Ge Li; Jing Chen; Jian-Ming Tan

    2007-01-01

    AIM:To ifivestigate the influence of heme oxygenase-1(HO-1)gene transfer on the viability and function of cultured rat islets in vitro.METHODS:Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion,and purified by discontinuous Ficoll density gradient centrifugation.Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene(Ad-HO-1)or enhanced green fluorescent protein gene(Ad-EGFP),and then cultured for seven days.Transfection was confirmed by fluorescence microscopy and Western blot.Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining.Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets.Stimulation index (SI)was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation.RESULTS:After seven days culture,the viability of cultured rat islets decreased significantly(92% ± 6% vs 52% ± 13%,P < 0.05),and glucose-stimulated insulin release also decreased significantly(6.47 ± 0.55 mIU/L/30IEQ vs 4.57 ± 0.40 mIU/L/30IEQ,14.93 ± 1.17mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ,P < 0.05).Transfection of rat Islets with adenoviral vectors at an MOI of 20 was efficient,and did not impair islet function.At 7 d post-transfection,the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% ± 15% vs 52% ± 13%,P < 0.05).There was no significant difference in insulin release upon low glucose stimulation(2.8 mmol/L)among Ad-HO-1 transfected group,Ad-EGFP transfected group,and control group(P > 0.05),while when stimulated by high glucose(16.7 mmol/L)solution,insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group,respectively(12.50 ± 2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ;12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ

  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. 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...

  14. Gene transfer of GLT-1, a glial glutamate transporter, into the spinal cord by recombinant adenovirus attenuates inflammatory and neuropathic pain in rats

    Nakagawa Takayuki

    2008-12-01

    Full Text Available Abstract Background The glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats. Results Intraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation. Conclusion These results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.

  15. Lifetime correction of genetic deficiency in mice with a single injection of helper-dependent adenoviral vector

    Kim, In-Hoo; Józkowicz, Alicja; Piedra, Pedro A.; Oka, Kazuhiro; Chan, Lawrence

    2001-01-01

    Ideally, somatic gene therapy should result in lifetime reversal of genetic deficiencies. However, to date, phenotypic correction of monogenic hyperlipidemia in mouse models by in vivo gene therapy has been short-lived and associated with substantial toxicity. We have developed a helper-dependent adenoviral vector (HD-Ad) containing the apolipoprotein (apo) E gene. A single i.v. injection of this vector completely and stably corrected the hypercholesterolemia in apoE-deficient mice, an effect...

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

    Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x1011 particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy

  1. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  2. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  3. 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...

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Inhibition of apoptosis reduces immunogeneic potential of adenoviral-treated syngeneic liver grafts.

    Puellmann, Kerstin; Beham, Alexander; Kienle, Klaus; Vogel, Mandy; Schlitt, Hans Juergen; Jauch, Karl Walter; Rentsch, Markus

    2006-11-27

    Effects of adenoviral therapy and reduced apoptosis on immune response were investigated in a rat liver transplantation model after prolonged ischemia-reperfusion. Liver donors were treated i.v. either with an adenoviral construct, expressing bcl-2, green-fluorescent-protein, or doxycyclin. Intrahepatic apoptosis was assessed by terminal transferase dUTP nick end labeling assay. The intrahepatic presence of CD4, CD8a, CD163, immunoglobulin (Ig)beta, tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO) was quantified by realtime polymerase chain reaction at 24 hours and seven days after transplantation. Bcl-2 expression abrogated the TNF-alpha elevation and reduced apoptosis of hepatocytes and sinusoidal endothelial cells as compared to advCMV green fluorescent protein. No effects on CD4, CD8a, CD163 and MPO expression were noticed in bcl-2 pretreated livers, whereas Igbeta was slightly enhanced compared to controls. Adenoviral infected liver grafts trigger an immune response but reduced apoptosis resulted in down-regulation of TNF-alpha. Thus, bcl-2 transfer might simultaneously reduce graft ischemia reperfusion injury and immunogenicity. PMID:17130789

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.)

  1. 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

  2. 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

  3. 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.

  4. 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...

  5. 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.

  6. 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

  7. 放射诱导调控腺病毒介导gfp报告基因在肿瘤细胞内的表达%The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation

    魏道严; 戴冰冰; 陈诗书; 王中和

    2001-01-01

    为建立放射诱导基因表达调控系统并用于肿瘤的基因治疗, 利用PCR技术克隆出放射诱导基因Egr-1基因启动子, 经测序证实后与报告基因gfp连接, 并利用新型、高效的细菌内同源重组腺病毒载体制备方法制备出重组腺病毒AdEgr-GFP。 感染腺病毒的肿瘤细胞给予不同剂量的γ射线照射, 体外采用FACS方法检测GFP的表达发现, 照射可明显提高GFP表达, 并呈剂量依赖性, Western印迹检测也显示类似的结果。 为进行体内实验, 瘤内注射AdEgr-GFP腺病毒后48 h, 肿瘤局部接受不同剂量的γ射线照射, 8 h后制备肿瘤组织标本用于分析GFP的表达。 肿瘤组织图像分析结果显示, γ射线照射可显著提高肿瘤组织中GFP的表达, 并呈剂量依赖性。 结果说明, 放射经Egr-1启动子可有效调控腺病毒介导gfp基因的肿瘤细胞内表达。%In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene. Then the pEgr-gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr-GFP by using a novel, high efficient method of homologous recombination in bacteria. After infection with AdEgr-GFP, MM45T.Li tumor cells were exposed to different doses of γ-irradiation from 0 Gy to 15 Gy in vitro. The percentage of GFP expression positive cells increased greatly in a dose-dependent manner as detected by FACS and Western blot analysis. For in vivo study, AdEgr-GFP were injected intratumorally, and tumor site received different doses of local γ-irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression. Tumor tissue image analysis revealed that γ-irradiation could markedly increase GFP expression in a dose-dependent manner as

  8. 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.

  9. 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

  10. 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.

  11. 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...

  12. Potential of Helper-Dependent Adenoviral Vectors in Modulating Airway Innate Immunity

    Rahul Kushwah; Huibi Cao; Jim Hu

    2007-01-01

    Innate immune responses form the first line of defense against foreign insults and recently significant advances have been made in our understanding of the initiation of innate immune response along with its ability to modulate inflammation. In airway diseases such as asthma, COPD and cystic fibrosis, over reacting of the airway innate immune responses leads to cytokine imbalance and airway remodeling or damage. Helper-dependent adenoviral vectors have the potential to deliver genes to modulate airway innate immune responses and have many advantages over its predecessors. However, there still are a few limitations that need to be addressed prior to their use in clinical applications.

  13. 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

  14. 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

  15. 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.

  16. Immunization Against the Transgene but not the TetON Switch Reduces Expression From Gutless Adenoviral Vectors in the Brain

    Xiong, Weidong; Candolfi, Marianela; Kroeger, Kurt M.; Puntel, Mariana; Mondkar, Sonali; Larocque, Daniel; Liu, Chunyan; Curtin, James F.; Palmer, Donna; Ng, Philip; Lowenstein, Pedro R; Castro, Maria G.

    2008-01-01

    Immune responses against vectors or encoded transgenes can impose limitations on gene therapy. We demonstrated that tetracycline-regulated high-capacity adenoviral vectors (HC-Ads) sustain regulated transgene expression in the brain even in the presence of systemic pre-existing immune responses against adenoviruses. In this study we assessed whether systemic pre-existing immune responses against the transgene products, i.e., β-Gal or the tetracycline-dependent (TetON) regulatory transcription...

  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 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

  18. 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

  19. 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

  20. 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 ...

  1. Adenovirus-p53 gene therapy in human nasopharyngeal carcinoma xenografts

    Background: One major challenge to human cancer gene therapy, is efficient delivery of the gene-vector complex. Methods and results: Using two distinct human nasopharyngeal carcinoma (NPC) models, we demonstrate that intra-tumoural (IT) administration of adenoviral-mediated wild-type p53 gene therapy (Ad-p53) caused no greater inhibition of tumour growth as compared to ionizing radiation (XRT) alone. Detailed histologic examination of tumour sections demonstrated that <15% of tumour cells were transduced by IT adv-β-gal. Conclusions: This report underscores the importance of developing gene transfer vectors, which can provide therapeutic levels of transgene expression efficiently in solid tumours

  2. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T;

    2004-01-01

    -expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists......We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures....... Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co...

  3. 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

  4. 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.

  5. 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

  6. 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...

  7. 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.

  8. A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors.

    Heger, Klaus; Kober, Maike; Rieß, David; Drees, Christoph; de Vries, Ingrid; Bertossi, Arianna; Roers, Axel; Sixt, Michael; Schmidt-Supprian, Marc

    2015-06-01

    Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology. PMID:25787118

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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...

  14. 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...

  15. Quality of the transgene-specific CD8+ T cell response induced by adenoviral vector immunization is critically influenced by virus dose and route of vaccination

    Holst, Peter Johannes; Ørskov, Cathrine; Thomsen, Allan Randrup;

    2010-01-01

    Adenoviral vectors have been widely used for experimental gene therapy and vaccination, yet there is a surprising lack of knowledge connecting the route and dose of adenovirus administration to the induced transgene-specific immune response. We have recently demonstrated polyfunctional CD8(+) T c...... effector functions, accumulated in the spleen. These findings indicate that the localization of the adenoviral inoculum and not the total Ag load determines the quality of the CD8(+) T cell response induced with adenoviral vaccines.......Adenoviral vectors have been widely used for experimental gene therapy and vaccination, yet there is a surprising lack of knowledge connecting the route and dose of adenovirus administration to the induced transgene-specific immune response. We have recently demonstrated polyfunctional CD8(+) T...... correlated positively with dissemination, whereas the functional capacity of the generated T cells correlated inversely with vector dissemination. A comparison of the immune response to s.c. or i.v. administration at moderate doses revealed that inoculation by both routes induced a transient peak of IFN...

  16. 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.

  17. 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...

  18. 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.

  19. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  20. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  1. 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...

  2. 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.

  3. 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.

  4. 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...

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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)

  14. 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...

  15. 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...

  16. 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...

  17. 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....

  18. 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...

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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...

  4. 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.)

  5. Antitumor Activity and Prolonged Expression from a TRAIL-Expressing Adenoviral Vector

    Jeongwu Lee

    2002-01-01

    Full Text Available Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL induces apoptosis in a variety of transformed cell lines, but generally spares most normal cells. Transduction by an adenoviral vector expressing human TRAIL cDNA (Ad.TRAIL-GFP resulted in both direct tumor cell killing as well as a potent bystander effect through presentation of TRAIL by transduced normal cells. Administration of Ad.TRAIL-GFP significantly prolonged survival of mice harboring either intracerebral glioblastomas or breast carcinoma-induced peritoneal carcinomatosis. Additionally, TRAIL induced prolonged transgene expression in normal tissue, presumably as a result of diminished immunemediated destruction of vector-transduced cells. Taken together, these data suggest that vector-mediated transduction of TRAIL may represent an effective strategy for cancer gene therapy.

  6. 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...

  7. 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...

  8. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H+/K+ ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H+/K+ ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H+/K+ ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H+/K+ ATPase which underpin the regulation of acid secretion

  9. 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...

  10. 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.