Novak, Thomas J. and Yoshimura, Fayth K. and Rothenberg, Ellen V. (1992) In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters. Molecular and Cellular Biology, 12 (4). pp. 1515-1527. ISSN 0270-7306 http://resolver.caltech.edu/CaltechAUTHORS:NOVmcb92
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We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.
|Additional Information:||Copyright © 1992 by the American Society for Microbiology. Received 29 August 1991/Accepted 6 January 1992 We thank Rochelle Diamond for advice on cell fractionation and flow cytometric data analysis and both Rochelle Diamond and Patrick Koen for excellent flow cytometry. We also thank Patricia White for quantitating some of the assays and for help assembling some of the figures. We are grateful to Kim Bottomly for her encouragement and gracious support of T.J.N. during part of this work; to James Allison and Nilabh Shastri for valuable cell lines; to Dorothy Lloyd for expert illustration; and to Cathy Blagg for tireless and excellent help in preparing the manuscript. This research was supported by PHS grants A119752 and CA39605 (to E.V.R.) and CA44166 (to F.K.Y.). Partial support for T.J.N. was provided by NIH postdoctoral training grant 5T32 A107019 and funds from the Howard Hughes Medical Institute.|
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|Deposited On:||27 Apr 2006|
|Last Modified:||26 Dec 2012 08:50|
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