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Retroviral Vectors (retroviral + vector)

Distribution by Scientific Domains

Life Sciences	65%
Medical Sciences	31%

Selected Abstracts

Retroviral vector silencing during iPS cell induction: An epigenetic beacon that signals distinct pluripotent states

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008
Akitsu Hotta
Abstract Retroviral vectors are transcriptionally silent in pluripotent stem cells. This feature has been potently applied in studies that reprogram somatic cells into induced pluripotent stem (iPS) cells. By delivering the four Yamanaka factors in retroviral vectors, high expression is obtained in fibroblasts to induce the pluripotent state. Partial reprogramming generates Class I iPS cells that express the viral transgenes and endogenous pluripotency genes. Full-reprogramming in Class II iPS cells silences the vectors as the endogenous genes maintain the pluripotent state. Thus, retroviral vector silencing serves as a beacon marking the fully reprogrammed pluripotent state. Here we review known silencer elements, and the histone modifying and DNA methylation pathways, that silence retroviral and lentiviral vectors in pluripotent stem cells. Both retroviral and lentiviral vectors are influenced by position effects and often exhibit variegated expression. The best vector designs facilitate full-reprogramming and subsequent retroviral silencing, which is required for directed-differentiation. Current retroviral reprogramming methods can be immediately applied to create patient-specific iPS cell models of human disease, however, future clinical applications will require novel chemical or other reprogramming methods that reduce or eliminate the integrated vector copy number load. Nevertheless, retroviral vectors will continue to play an important role in genetically correcting patient iPS cell models. We anticipate that novel pluripotent-specific reporter vectors will select for isolation of high quality human iPS cell lines, and select against undifferentiated pluripotent cells during regenerative medicine to prevent teratoma formation after transplantation. J. Cell. Biochem. 105: 940,948, 2008. © 2008 Wiley-Liss, Inc. [source]

Scaleable purification process for gene therapy retroviral vectors

THE JOURNAL OF GENE MEDICINE, Issue 4 2007
Teresa Rodrigues
Abstract Background Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology. Methods A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 µm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold. Results Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77 ± 11%). Up-scaling of the process resulted in high titer vector preparations, 3.2 × 108 infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination. Conclusions The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies. Copyright © 2007 John Wiley & Sons, Ltd. [source]

In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy

THE JOURNAL OF GENE MEDICINE, Issue 3 2005
Georgia Mavria
Abstract Background Retroviral vectors are suitable for targeting endothelial cells in the tumour neovasculature because of their intrinsic selectivity for proliferating cells. Previously, we inserted regulatory elements of the endothelial-specific prepro-endothelin-1 (ppET1) promoter in retroviral vectors to generate high-titre, replication-defective recombinant retroviruses that restricted gene expression to the vascular compartment of tumours. Methods A retroviral vector was generated in which expression of herpes simplex virus thymidine kinase (HSV-TK) was transcriptionally restricted to endothelial cells, under the control of a hybrid ppET-1 LTR. Xenograft tumour models were used to determine the efficacy of targeting HSV-TK to the tumour vasculature. Subsequently, vascular-targeted gene therapy was combined with chemotherapeutic agents. Results Breast or colorectal xenograft tumour growth was reduced and survival was increased in response to ganciclovir treatment. Treatment resulted in widespread vascular disruption and tumour cell apoptosis. In colorectal tumours, combination with irinotecan, a cytotoxic drug used to treat colorectal cancer, significantly increased survival compared to drug alone. No beneficial effect on survival was observed when combined with cisplatin, a cytotoxic drug not in clinical use for this tumour type. On the basis of their relative efficacies in vitro against tumour and endothelial cells, co-operativity with irinotecan likely derives from additionally targeting the peripheral tumour cells that survive the anti-vascular treatment. Conclusions We show that the ppET1-targeted vector is efficacious for therapeutic gene expression in vivo, validating a strategy targeted to tumour vasculature, and demonstrate that vascular targeting combined with appropriate chemotherapy is more effective than either therapy alone. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Protection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbones

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2001
Michael Jansen
Abstract: Overexpression of O6 -methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6 -alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6 -alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8,12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [source]

TCR-, chains derived from peripheral ,, T cells can take part in ,, T-cell development

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2008
Nabil Bosco
Abstract Between 10 and 20% of the peripheral ,, T cells express cytoplasmic TCR-, proteins, but whether such TCR-, chains can partake in ,, T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3,-deficient mice with Pax5-TCR-, deficient proB cells expressing, via a retroviral vector, TCR-, chains from either peripheral ,, or ,, T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15×106 cells), contained few ,, T but no ,, T cells. In contrast, thymi from mice receiving proB cells containing ,, or ,, T-cell-derived TCR-, chains contained 80,130×106 cells, and showed a normal CD4, CD8 and ,, TCR expression pattern. However, regardless of the source of TCR-, chain, reconstituted mice rapidly showed signs of autoimmunity dying 5,15,wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-, chains from ,, T cells can efficiently take part in ,, T-cell development. The implications of these findings for ,, T-cell development will be discussed. [source]

Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model

EXPERIMENTAL DERMATOLOGY, Issue 11 2006
Lina Elzaouk
Abstract:, Mesenchymal stem cells (MSCs) represent a new tool for delivery of therapeutic agents to tumor cells. In this study, we have evaluated the anti-tumor activity of human MSCs stably transduced with a retroviral vector expressing the cytokine interleukin-12 (IL-12) in a mouse melanoma model. Application of MSC(IL-12) but not control MSCs strongly reduced the formation of lung metastases of B16F10 melanoma cells. The activity of the MSC(IL-12) cells was dependent on the presence of natural killer (NK) cells in this experimental setting. Further, MSC(IL-12) cells elicited a pronounced retardation of tumor growth and led to prolonged survival when injected into established subcutaneous melanoma in a therapeutic regimen. The therapeutic effect of the MSC(IL-12) was in part mediated by CD8+ T cells, while NK cells and CD4+ T cells appeared to play a minor role. The anti-tumor effect of MSC(IL-12) cells was of similar efficiency as observed for application of naked plasmid DNA encoding IL-12. The presented data demonstrate that these two different strategies can induce a similar therapeutic anti-tumor efficacy in the mouse melanoma tumor model. [source]

Retroviral labeling of Schwann cells: In vitro characterization and in vivo transplantation to improve peripheral nerve regeneration

GLIA, Issue 1 2001
Afshin Mosahebi
Abstract Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ -labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo. GLIA 34:8,17, 2001. © 2001 Wiley-Liss, Inc. [source]

Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2004
Basem M Abdallah
Abstract Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPAR,2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor ,1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes. [source]

Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2001
Isabelle A. Franceschini
Abstract Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells. J. Neurosci. Res. 65:208,219, 2001. © 2001 Wiley-Liss, Inc. [source]

Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat

THE JOURNAL OF GENE MEDICINE, Issue 3 2008
Charles H. Rundle
Abstract Background An in vivo gene therapy strategy was developed to accelerate bone fracture repair. Methods Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3,-untranslated region and to improve protein translation. Results In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E2 and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed. Conclusions This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair. Copyright © 2007 John Wiley & Sons, Ltd. [source]

In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL)

THE JOURNAL OF GENE MEDICINE, Issue 4 2004
Hiromichi Katakura
Abstract Background Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. Methods A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. Results We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. Conclusions PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice

THE JOURNAL OF GENE MEDICINE, Issue 1 2004
Xiao S. Zhang
Abstract Background Bone loss in osteoporosis is caused by an imbalance between resorption and formation on endosteal surfaces of trabecular and cortical bone. We investigated the feasibility of increasing endosteal bone formation in mice by ex vivo gene therapy with bone marrow stromal cells (MSCs) transduced with a MLV-based retroviral vector to express human bone morphogenetic protein 4 (BMP4). Methods We assessed two approaches for administering transduced MSCs. ,-Galactosidase (,-Gal) transduced C57BL/6J mouse MSCs were injected intravenously via tail vein or directly injected into the femoral bone marrow cavity of non-marrow-ablated syngenic recipient mice and bone marrow cavity engraftment was assessed. BMP4- or ,-Gal-transduced cells were injected into the femoral bone marrow cavity and effects on bone were evaluated by X-ray, peripheral quantitative computed tomography (pQCT), and histology. Results After tail-vein injection less than 20% of recipient mice contained ,-Gal-positive donor cells in femur, humerus or vertebra marrow cavities combined, and in these mice only 0.02,0.29% of injected cells were present in the bone marrow. In contrast, direct intramedullary injection was always successful and an average of 2% of injected cells were present in the injected femur marrow cavity 24 hours after injection. Numbers of donor cells decreased over the next 14 days. Intramedullary injection of BMP4-transduced MSCs induced bone formation. Trabecular bone mineral density (BMD) determined by pQCT increased 20.5% at 14 days and total BMD increased 6.5% at 14 days and 10.4% at 56 days. Conclusions The present findings support the feasibility of using ex vivo MSC-based retroviral gene therapy to induce relatively sustained new bone formation, with normal histological appearance, at endosteal bone sites. Copyright © 2004 John Wiley & Sons, Ltd. [source]

In vivo expansion of transduced murine hematopoietic cells with a selective amplifier gene

THE JOURNAL OF GENE MEDICINE, Issue 3 2003
Akihiro Kume
Abstract Background Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed ,selective amplifier genes' (SAGs) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGs conferred estrogen-responsive growth on murine hematopoietic progenitors. Methods We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP+ cells were enumerated. Results The challenge induced a significant increase in EGFP+ leukocytes (21 ± 4% to 27 ± 5%), while EGFP+ cells decreased in untreated animals (21 ± 5% to 10 ± 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP+ cells (16 ± 4% to 35 ± 3%), contrasting to a decrease in controls (22 ± 4% to 12 ± 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP+ cells in granulocytes and monocytes following Tm administration. Conclusions Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Blocking vascular endothelial growth factor with soluble Flt-1 improves the chondrogenic potential of mouse skeletal muscle,derived stem cells

ARTHRITIS & RHEUMATISM, Issue 1 2009
Seiji Kubo
Objective To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle. Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real-time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP-4) were coimplanted with MDSCs transduced to express either VEGF or sFlt-1 (a VEGF antagonist) to provide a gain- and loss-of-function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt-1,treated) pellet cultures, but quantitative PCR revealed that sFlt-1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP-4 and transforming growth factor ,3 (TGF,3). In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt-1 improved the MDSC-mediated repair of articular cartilage, compared with BMP-4 alone. Conclusion Soluble Flt-1 gene therapy improved the BMP-4, and TGF,3-induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage. [source]

Problem-solving test: Telomere replication

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010
József Szeberényi
Terms to be familiar with before you start to solve the test: DNA replication, template, primer, linear DNA, antiparallel orientation, telomere, origin of replication, chromatid, replicon, short tandem repeats, Okazaki fragments, leading strand, lagging strand, ribozyme, promoter, enhancer, terminal transferase, DNA polymerases, reverse transcriptase, RNA polymerase, topoisomerase, retroviral vector, Southern blotting, restriction endonuclease. [source]

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010
Shuohao Huang
Abstract Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717,729. © 2010 Wiley Periodicals, Inc. [source]

Retrovirus vector-mediated gene transfer into the chick optic vesicle by in ovo electroporation

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008
Hiraki Sakuta
Owing to its external position in the embryo, the chick eye has been used as a readily accessible model for studying the molecular mechanisms behind the patterning of the central nervous system. Although methods of genetic analysis have not been established as in the mouse, the chick is convenient for analyzing the functions of genes by in ovo electroporation of retroviral vectors. In this review, we describe the retroviral vector-mediated transfer of genes into the chick optic vesicle by in ovo electroporation. A rapid, efficient, and sustained expression of transgenes is achieved by this approach. [source]

Protection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbones

Haematopoietic progenitor cells from the common marmoset as targets of gene transduction by retroviral and adenoviral vectors

EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2001
Hitoshi Hibino
Abstract: To establish a new non-human primate model for human cytokine and gene therapy, we characterized lymphocytes and haematopoietic progenitor cells of the small New World monkey, the common marmoset. We first assessed the reactions of marmoset bone marrow (BM) and peripheral blood (PB) cells to mouse anti-human monoclonal antibodies (mAbs) for the purpose of isolating marmoset lymphocytes and haematopoietic progenitor cells. Both cell fractions stained with CD4 and CD8 mAbs were identified as lymphocytes by cell proliferation assay and morphological examination. Myeloid-specific mAbs such as CD14 and CD33 did not react with marmoset BM and PB cells. No available CD34 and c-kit mAbs could be used to purify the marmoset haematopoietic progenitor cells. Furthermore, we studied the in vitro transduction of the bacterial ,-galactosidase (LacZ) gene into CFU-GM derived from marmoset BM using retroviral and adenoviral vectors. The transduction efficiency was increased by using a mixed culture system consisting of marmoset BM stromal cells and retroviral producer cells. It was also possible to transduce LacZ gene into marmoset haematopoietic progenitor cells with adenoviral vectors as well as retroviral vectors. The percentage of adenovirally transduced LacZ-positive clusters was 15% at day 4 (multiplicity of infection=200), but only 1,2% at day 14. The differential use of viral vector systems is to be recommended in targeting different diseases. Our results suggested that marmoset BM progenitor cells were available to examine the transduction efficiency of various viral vectors in vitro. [source]

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats

HAEMOPHILIA, Issue 3 2003
A. Van Damme
No abstract is available for this article. [source]

Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells

HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 2 2008
William C. Spanos MD
Abstract Background Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60% of sampled specimens. Methods To understand better the viral genes required to transform human tonsil epithelial cells (HTEC), we isolated HTEC's and transduced them with retroviral vectors containing HPV16 E6 and E7. Results Immortalization and anchorage-independent growth of HTEC's only occurred with expression of E6 and E7 with resultant degradation of p53. However, cells expressing E6 lacking the PSD-95/disc-large/Zo-1 (PDZ) motif did not immortalize or grow anchorage independent. Telomerase activity and degradation of p53 were similar for wild-type and mutant E6. Conclusion The mechanism of oncogenic transformation by E6 in HTEC's is dependent on the PDZ binding motif. Identification of pathways affected by the interaction of E6 and PDZ domain containing proteins will further our understanding of how HPV causes HNSCC and will provide potential therapeutic targets. © 2007 Wiley Periodicals, Inc. Head Neck, 2008 [source]

Cellular responses in experimental liver injury: Possible cellular origins of regenerative stem-like progenitor cells,

HEPATOLOGY, Issue 5 2005
William B. Coleman Ph.D.
Background/Aims Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes. Methods Mature hepatocytes were genetically labeled using retroviral vectors harboring the ,-galactosidase gene. After labeling, retrorsine was administered to rats followed by partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later. Results We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed ,-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of ,-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice. Conclusions The constant proportion of ,-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury. [source]

Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2006
Tian-Fang Li
Abstract Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF-, signaling factors, but BMP-related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3,/, chondrocytes. BMP signaling is increased in TGF-, deficiency and is required for accelerated chondrocyte maturation. Introduction: Disruption of TGF-, signaling results in accelerated chondrocyte maturation and leads to postnatal dwarfism and premature osteoarthritis. The mechanisms involved in this process were studied using in vitro murine chondrocyte cultures. Materials and Methods: Primary chondrocytes were isolated from the sterna of neonatal wildtype and Smad3,/, mice. Expressions of maturational markers, as well as genes involved in TGF-, and BMP signaling were examined. Chondrocytes were treated with TGF-, and BMP-2, and effects on maturation-related genes and BMP/TGF-, responsive reporters were examined. Recombinant noggin or retroviral vectors expressing Smad2 or Smad3 were added to the cultures. Results: Expression of colX and other maturational markers was markedly increased in Smad3,/, chondrocytes. Smad3,/, chondrocytes lacked compensatory increases in Smad2, Smad4, TGFRII, Sno, or Smurf2 and had reduced expression of TGF - ,1 and TGFRI. In contrast, Smad1, Smad5, BMP2, and BMP6 expression was increased, suggesting a shift from TGF-, toward BMP signaling. In Smad3,/, chondrocytes, alternative TGF-, signaling pathways remained responsive, as shown by luciferase assays. These non-Smad3-dependent TGF-, pathways reduced colX expression and alkaline phosphatase activity in TGF-,-treated Smad3,/, cultures, but only partially. In contrast, Smad3,/, chondrocytes were more responsive to BMP-2 treatment and had increased colX expression, phosphoSmads 1, 5, and 8 levels, and luciferase reporter activity. Overexpression of both Smad2 and Smad3 blocked spontaneous maturation in Smad3-deficient chondrocytes. Maturation was also abrogated by the addition of noggin, an extracellular BMP inhibitor. Conclusions: These findings show a key role for BMP signaling during the chondrocyte maturation, occurring with loss of TGF-, signaling with important implications for osteoarthritis and cartilage diseases. [source]

Retroviral vector silencing during iPS cell induction: An epigenetic beacon that signals distinct pluripotent states

Scaleable purification process for gene therapy retroviral vectors

In vivo efficacy of HSV-TK transcriptionally targeted to the tumour vasculature is augmented by combination with cytotoxic chemotherapy

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L -lysine) block copolymer (PEG-PLL)

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector

Mathematical model of the rate-limiting steps for retrovirus-mediated gene transfer into mammalian cells

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010
Venkata S. Tayi
Abstract A quantitative understanding of the process of retrovirus-mediated gene transfer into mammalian cells should assist the design and optimization of transduction protocols. We present a mathematical model of the process that incorporates the essential rate-limiting transduction steps including diffusion, convection and decay of viral vectors, their binding at the cell surface and entry into the cell cytoplasm, reverse transcription of uncoated RNA to form DNA intermediates, transport of the latter through the cytosol to the cell nucleus and, finally, nuclear import and integration of the delivered DNA into the target cell genome. Cell and virus population balances are used to account for the kinetics of multiple vector infections which influence the transduction efficiency and govern the integrated copy number. The mathematical model is validated using gibbon ape leukemia virus envelope pseudotyped retroviral vectors and K562 target cells. Viral intermediate complexes derived from the internalized retroviral vectors are found to remain stable inside the K562 cells and the cytoplasmic trafficking time is consistent with the time scale for retrovirus uncoating, reverse transcription and transport to the cell nucleus. The model predictions of transduction efficiency and integrated copy number agree well with experimental data for both static (i.e., standard gravity) and centrifugation-based gene transfer protocols. The formulation of the model can also be applied to transduction protocols involving lenti- or foamy-viruses and so should prove to be useful for the optimization of several types of gene transfer processes. Biotechnol. Bioeng. 2010;105: 195,209. © 2009 Wiley Periodicals, Inc. [source]