Transfer of activation-dependent gene expression into T cell lines by recombinant adeno-associated virus.
We examined the ability of recombinant adeno-associated virus (rAAV) to transfer regulated gene expression into T cell lines. An AAV-based vector containing the neomycin resistance gene and expressing the firefly luciferase (luc) gene under the regulatory control of the interleukin 2 promoter (pAAV-luc) was generated and adenovirus-free rAAV (rAAV-luc) was produced from this vector. Transfection of pAAV-luc into the human T cell line Jurkat resulted in luciferase expression while infection of Jurkat T cells with rAAV-luc resulted in significant luciferase expression only after selection for neomycin-resistant cells. Long-term growth of transduced Jurkat T cells showed that there was no detectable constitutive expression of luciferase and that luciferase gene expression remained inducible for at least 180 days. Luciferase expression was activated by PMA and ionomycin and by anti-CD3 antibodies and was inhibited by cyclosporin A. Examination of G418-resistant clones showed that rAAV-luc had integrated into the host chromosomes but that some of the clones lost some of the transferred DNA or lost expression from the transferred DNA. These results indicate that rAAV can transfer and integrate regulated gene expression into T cell lines but that the transferred genetic material may be lost or its expression may be silenced over time.
Zhang
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PX
Yale Child Health Research Center, Yale University School of Medicine, New Haven, CT 06520-8081, USA.
Fuleihan
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RL
D000229:Dependovirus / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics; D008213:Lymphocyte Activation; D009355:Neomycin; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors
D007376:Interleukin-2; D008156:Luciferases; D009355:Neomycin