No abstract available.
Humans* ; Retroviridae*

Interleukin 12 (IL-12), a heterodimeric cytokine, promotes an effective antitumor response against tumors of various histological types when delivered systemically as a protein or locally by gene transfer. We investigated parameters that influenced the effectiveness of IL-12 retroviral-mediated gene therapy of cancer in animals using the murine breast cancer line TS/A. Syngeneic fibroblasts (TIB80), stably transduced with a retrovirus expressing murine IL-12, were used for peritumoral injection. Injection of fibroblasts into established tumors resulted in complete regression of tumor in 40 % of animals in a dose dependent manner when treated on day 4, and 20 % when treated on day 8. Significant inhibition of growth of day 21 and day 40 tumors was observed following peritumoral injection of IL-12-expressing fibroblasts in a dose-dependent manner. Delivery of IL-12 by syngeneic fibroblasts at a tumor site is effective in eradicating established, weakly immunogenic TS/A tumors.
Animals ; Breast Neoplasms ; Fibroblasts ; Genetic Therapy* ; Interleukin-12* ; Retroviridae

Background : Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs (suicide genes) into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase (HSV-tk) gene followed by administration of the antiviral drug ganciclovir. METHODS: The LNC/tK vector was transfered in vitro into mouse Neuro 2a cell lines (ATCC) and the transduced cell lines were selected in G-418, 500microgram/ml, for 14 days. Onex104 cells were cultured in 96 well culture plates in increasing concentrations of ganciclovir for 72 hours. The sesitivity to ganciclivir of these HSV-tk transduced, G-418 selected cells was measured with MTT assay RESULTS: The survival of HSV-tk transduced 1x104 neuro 2a cell lines is 103+/-3.5%, 68+/-4.2%, 54+/-3.8%, 17+/-2.6%, 13+/-3.1% at the concentration of 0, 0.1, 1.0, 10, 20microgram/ml ganciclovir, respectively. And the survival of HSV-tk not transduced 1x104 neuro 2a cell lines is 100+/-4.5%, 97+/-5.6%, 104+/-3.5%, 106+/-3.8%, 101+/-4.2%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Animals ; Cell Line* ; DNA ; Ganciclovir ; Mice ; Neuroblastoma* ; Phosphotransferases ; Retroviridae ; Zidovudine*

Background : Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs (suicide genes) into proliferating tumor may be used to treat cancer. We investigated the efficacy of in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase (HSV-tk) gene followed by administration of the antiviral drug ganciclovir. METHODS: The LNC/tK vector was transfered in vitro into mouse Neuro 2a cell lines (ATCC) and the transduced cell lines were selected in G-418, 500microgram/ml, for 14 days. Onex104 cells were cultured in 96 well culture plates in increasing concentrations of ganciclovir for 72 hours. The sesitivity to ganciclivir of these HSV-tk transduced, G-418 selected cells was measured with MTT assay RESULTS: The survival of HSV-tk transduced 1x104 neuro 2a cell lines is 103+/-3.5%, 68+/-4.2%, 54+/-3.8%, 17+/-2.6%, 13+/-3.1% at the concentration of 0, 0.1, 1.0, 10, 20microgram/ml ganciclovir, respectively. And the survival of HSV-tk not transduced 1x104 neuro 2a cell lines is 100+/-4.5%, 97+/-5.6%, 104+/-3.5%, 106+/-3.8%, 101+/-4.2%. CONCLUSION: We concluded that in vitro transduction of neuroblastoma cell with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir is very effective.
Animals ; Cell Line* ; DNA ; Ganciclovir ; Mice ; Neuroblastoma* ; Phosphotransferases ; Retroviridae ; Zidovudine*

Human leukemia is closely associated with various genetic alterations such as chromosomal translocations and gene mutations. The use of retroviral transduction/bone marrow transplantation mouse model harboring these genetic abnormalities has been critical in understanding the molecular pathogenesis of leukemia and exploring new therapeutic target. Additional genetic events are verified to cooperate with fusion genes resulting from chromosomal translocations in acute myeloid leukemia (AML) to develop a leukemic phenotype in mice, such as C-KIT N822K with AML1-ETO, FLT3-ITD with PML-RARα, Meis1 with NUP98-HOX, and Cdx4 with MLL-AF9. Mouse model shows that BCR/ABL fusion gene induces chronic myeloid leukemia (CML), and suggests that GATA-2 L359V and high expression of Hes1 are key molecules in acute myeloid transformation of CML. Furthermore, combination therapy with Imatinib and arsenic sulfide for CML mice exerts more profound therapeutic effects than either drug as a single agent. This review focuses the recent progress and application of retroviral-mediated mouse models of myeloid leukemia, and discusses some factors influencing the mouse model establishment, including retroviral construction, retrovirus titer and hematopoietic microenvironment.
Animals ; Disease Models, Animal ; Leukemia, Myeloid ; genetics ; Mice ; Retroviridae ; genetics

Foamy virus can establish lifelong persistent infection in mammal hosts without inducing diseases. Such special characteristic stimulates the interests of researchers. As reported, the accessory protein Bet of foamy virus could regulate the gene expression and infection cycle of foamy virus and take part in the generation of chronic viral infection. And also, Bet might prevent the host cellular defense factor APO-BEC3 from interfering the replication of virus and play a role in maintaining viral persistent infection. In order to elucidate the roles of Bet in the foamy virus replication and infection, this review summarized the research progress of Bet protein reported in recent years.
Animals ; Gene Expression Regulation, Viral ; Humans ; Retroviridae Infections ; immunology ; virology ; Retroviridae Proteins ; genetics ; metabolism ; Spumavirus ; genetics ; metabolism

OBJECTIVETo explore use of retroviral vector in gene therapy of hepatitis B.

METHODSThe recombinant vector Plxsn-HBs was constructed by inserting HBV S gene into pLXSN. The pseudovirus, which was produced from PA317 after transferring with pLXSN-HBs by electroporation, were frozen at different temperature. The activities of the pseudovirus to infect eukaryotic cells and express antigen were determined by comparing the numbers of G418-resistant clones and assaying HBsAg in the supernatant of the cells with ELISA after infection HepG2, NIH3T3 and 293 cells.

RESULTSIt was hard to find changes in HBsAg amount at different intervals and different temperatures. G418 resistant clones, however, were variable. When frozen at -20 degrees C, the numbers of clones were half less than that of the beginning after 6 months, few clones were formed after 12 months, and no clone was found after 24 months. When frozen at -40 degrees C, the numbers of clones were 121, 332 and 89 42, 137 and 43 for HepG2, NIH3T3 and 293 cell lines at 12 and 24 months, respectively. When frozen at -70 degrees C, the numbers of clones were 159 463 and 112 for HepG2, NIH3T3 and 293 cell lines at 24 months, respectively. There was no statistical difference compared to that of zero months.

CONCLUSIONSThe activity of the peseudovirus to infect eukaryotic cells and expressed antigen was not changed after 2 years frozen at -70 degrees C.

This study was purposed to construct the recombinant retroviral vector containing human homeobox gene hoxA10 and to establish the packaging cell lines which stably produce viruses. The whole coding region of hoxA10 gene was amplified by PCR and inserted into the retroviral vector MSCVneo. The recombinant vector was identified by DNA sequencing. The recombinant and control retroviral vectors were transfected into the packaging cell line PT67 by liposome Lipofectamine(TM) 2000. These cell lines stably producing retrovirus were isolated following G418 selection. The viral suspension was harvested and the viral titer was determined by NIH3T3 cells. The results showed that the recombinant retroviral vector was proved to encode hoxA10 genes by sequencing. The cell lines efficiently producing virus were screened by G418 and designated as PT67/MSCVneo and PT67/MSCVneo-hoxA10. The titers of them were 5 x 10(5) CFU/ml and 4 x 10(4) CFU/ml respectively. It is concluded that the recombinant retroviral vector containing homeobox gene hoxA10 and the stably packaging cell lines which efficiently and correctly produce viruses are successfully constructed, which provides a basis for further exploration of the hoxA10 gene function in the regulation of hematopoiesis.
Cell Line ; Genes, Homeobox ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Retroviridae ; genetics ; Transfection

PURPOSE: We tried to assess the optimal conditions to improve low transduction efficiency and their effect on target cells. METHODS: Cultured NIH 3T3 cells were incubated with retroviral vectors bearing an enhanced green fluorescent protein (eGFP) gene. We varied the ratio of viral vectors to target cells (1:1-1:8) and the number of transfections (x1, x2), and compared transduction efficiencies. Also, the effects of polybrene on transduction efficiency and viability of target cells were assessed. Transduction of the eGFP gene was evaluated by observing NIH 3T3 cells under a fluorescence microscope and efficiencies were measured by the percentage of eGFP positive cells using FACscan. RESULTS: As the ratio of retroviral vectors to target cells increased, transduction efficiency was greatly improved, from 7% (1:1) to 38% (1:4). However, transduction efficiency did not increase any more when the ratio increased from 1:4 to 1:8. Cells transfected twice showed higher transduction efficiencies than cells transfected once, at a ratio of 1:8. The eGFP gene transduced to NIH 3T3 cells sustained its expression during repeated passages. However, after the third passage (day 9), the percentage of eGFP positive cells began to decline. The degree of this decline in eGFP expression was lower in cells transfected twice than in cells transfected once (P<0.05). The addition of polybrene did not have any toxic effect on NIH 3T3 cells and greatly increased transduction efficiency (P=0.007). In addition to vector component, transduction efficiency was very sensitive to culture confluence. Cells cultured and transfected in 24-well plate showed higher transduction efficiency, although cells cultured in 6- well plate proliferated more (P=0.024). CONCLUSION: Our data could be used as a basis for retrovirus-based gene therapy. Further study will follow using human cells as target cells.
Fluorescence ; Genetic Therapy ; Hexadimethrine Bromide ; Humans ; NIH 3T3 Cells* ; Retroviridae ; Transfection ; Zidovudine

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are. focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer, To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of leo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.
Cell Line ; Eukaryotic Cells ; Helper Viruses ; Product Packaging ; Retroviridae ; Transfection ; Zidovudine*