In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters.
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.
Novak
T J
TJ
Division of Biology, California Institute of Technology, Pasadena 91125.
Yoshimura
F K
FK
Rothenberg
E V
EV
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005786:Gene Expression Regulation; D005814:Genes, Viral / Q000235:genetics*; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000235:genetics; D008213:Lymphocyte Activation / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D013154:Spleen / Q000166:cytology; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology; D014162:Transfection
D006133:Growth Substances; D007376:Interleukin-2; D015500:Chloramphenicol O-Acetyltransferase