Wan Y Y, Leon R P, Marks R, Cham C M, Schaack J, Gajewski T F, DeGregori J
Departments of Biochemistry and Molecular Genetics and Pediatrics, and Microbiology, Program in Molecular Biology, University of Colorado Health Sciences Center, BRB802, 4200 East Ninth Avenue, Denver, CO 80262, USA.
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13784-9. doi: 10.1073/pnas.250356297.
The inability to easily and efficiently introduce genes into primary T cells has hampered the investigation of the pathways controlling T cell fate. To enable adenoviral-mediated gene transfer into normal naive T cells, transgenic (Tg) mice expressing the coxsackie/adenovirus receptor (CAR) in their T cell compartment were constructed. Whereas naive T cells are resistant to adenoviral infection, Tg expression of CAR on T cells greatly facilitates adenoviral-mediated gene expression ex vivo, in vivo, and in differentiated T helper cells. Thus we have developed a technology for efficient gene delivery to naive T cells. By using adenoviral vectors encoding specific inhibitors, we show that G1 cyclin-dependent kinase, NF-kappaB, and caspase activities are required for the proliferation of primary T cells. In addition, by expressing Bcl-x(L) protein at a level that closely approximates mitogen-induced levels, we demonstrate that Bcl-x(L) expression is sufficient to account for mitogen-mediated survival of primary T cells. Thus, adenoviral-mediated gene delivery to CAR Tg T cells should be useful for the analysis of many genes controlling T cell fate.
难以轻松且高效地将基因导入原代T细胞阻碍了对控制T细胞命运途径的研究。为了实现腺病毒介导的基因转移至正常的初始T细胞,构建了在其T细胞区室中表达柯萨奇病毒/腺病毒受体(CAR)的转基因(Tg)小鼠。虽然初始T细胞对腺病毒感染具有抗性,但T细胞上CAR的转基因表达极大地促进了腺病毒介导的基因在体外、体内以及分化的辅助性T细胞中的表达。因此,我们开发了一种向初始T细胞高效递送基因的技术。通过使用编码特异性抑制剂的腺病毒载体,我们表明G1期细胞周期蛋白依赖性激酶、核因子κB和半胱天冬酶活性是原代T细胞增殖所必需的。此外,通过以与促有丝分裂原诱导水平相近的水平表达Bcl-x(L)蛋白,我们证明Bcl-x(L)表达足以解释促有丝分裂原介导的原代T细胞存活。因此,腺病毒介导的基因递送至CAR转基因T细胞对于分析许多控制T细胞命运的基因应该是有用的。