Abstract
A bicistronic human immunodeficiency virus type 1 (HIV-1)-based vector is described in which the expression of a selectable marker and a second gene of interest are forcibly coupled by means of an internal ribosome entry site. The vector provides high-level expression of the coselected gene in approximately 90% of transduced cells and has been used to express an endoplasmic reticulum-targeted single-chain antibody (intrabody) directed against a subunit of the interleukin-2 receptor, IL-2Rα. In the established T cell line Kit225 and also in primary human T cells stably transduced with the intrabody vector, the cell surface expression of IL-2Rα could be reduced to a low or unde- tectable level. Responsiveness to IL-2 was reduced 10-fold in the IL-2Rα-negative cells, consistent with a lack of high-affinity IL-2 receptors. Pseudotyping of the HIV-1 core with the vesicular stomatitis virus G protein improved particle stability by two- to three-fold and enhanced vector entry into established T cell lines up to 230-fold. Vector entry into primary human T cells was most efficient when the amphotropic murine leukemia virus envelope was used. The forced, high-expression capability of the bicistronic vector, together with the capacity of HIV-1 vectors to infect nondividing cells, make this an attractive tool for the genetic manipulation of primary cell types.
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Richardson, J., Hofmann, W., Sodroski, J. et al. Intrabody-mediated knockout of the high-affinity IL-2 receptor in primary human T cells using a bicistronic lentivirus vector. Gene Ther 5, 635–644 (1998). https://doi.org/10.1038/sj.gt.3300644
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DOI: https://doi.org/10.1038/sj.gt.3300644
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