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- W2086489348 abstract "One of the principal mechanisms by which the immune system fails to reject a tumor is a lack of cytotoxic response activation. In order to circumvent this problem, we have genetically engineered murine colorectal carcinoma cells to actively express the IL-2 gene, introduced using three different retroviral vectors (DCTK-IL2, LNCX-IL2, LXSN-IL2). These three vectors also carried the neoR gene, which confers resistance to in vitro treatment with a neomycin analog (G418). We evaluated the effect of different selection methods on the final gene expression. Previous experiences demonstrated that, out of the three retroviral vectors with the IL-2 gene, one of them (DCTK-IL2) was very effective in stimulating a systemic specific antitumor response, but an inappropriate local response against the immunization dose. In order to control the growth of tumor cells transduced with the DCTK-IL2 vector injected as active immunization, these cells were further transduced with a retroviral vector carrying the neo gene and a “suicidal” gene, which codifies for the HSVTK. For these cells with a double transduction, we designed two selection methods (initially with high doses of G418 or HAT followed by G418) in order to identify the subclones which had the most favorable expression of both the IL-2 and HSVTK genes. IL-2 production increases both with increasing in vitro concentrations, and with duration of G418 selection. Assayed with a commercial IL-2 ELISA, the DCTK-IL2 vector produced the lowest 1L-2 levels in vitro (2 U/10 6 /24 h), whereas the other two retroviral vectors produced similar levels of IL-2 (18 U/10 6 /24 h). Cells with a double transduction were able to successfully tolerate higher levels of G418 in the culture (up to 1.6 mg/ml) due to the presence of two copies of the neoR gene. Both methods of selection of these cells (high dose G418 or HAT) produced cell populations sensible to GCV in vitro . We obtained 55 subclones from different moments of the ongoing cell selection, and the clones with most favorable expression of both genes (IL-2 and HSVTK) were expanded for further in vivo studies. In conclusion, we have shown that both the level and duration of selection of retrovirally transduced cells have an important impact on gene expression, and that it is possible to select cells with a double transduction with two vectors carrying the neoR gene. The most favourable cell clones will be used as active antitumoral immunization (vaccines) in further gene therapy experimental studies in Balb/c mice." @default.
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- W2086489348 date "1995-11-01" @default.
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- W2086489348 title "1277 Transduction and selection of murine tumor cells engineered to express the interleukin 2 (IL-2) and herpes simplex virus thymidine kinase (HSVTK) genes" @default.
- W2086489348 doi "https://doi.org/10.1016/0959-8049(95)96523-g" @default.
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