Goffinet Christine, Keppler Oliver T
Department of Virology, University of Heidelberg, Heidelberg, Germany.
FASEB J. 2006 Mar;20(3):500-2. doi: 10.1096/fj.05-4651fje. Epub 2006 Jan 9.
A rapid method for efficient gene delivery into primary rodent lymphocytes would greatly facilitate the study of signaling and metabolic pathways in untransformed hematopoietic cells as well as the validation of gene expression and targeting strategies before the generation of knockout or knock-down animals. Here, we report that species-adapted nucleofection procedures combined with optimized cultivation conditions render proliferating primary T cells, B cells, and natural killer cells from widely used rat and mouse strains susceptible to high-level gene delivery. As a result, transgene expression levels were enhanced approximately 10- to 370-fold over established protocols. The effectiveness of the nucleofection approach for functional analyses was demonstrated by specific down-regulation of CD4 cell surface molecules by either transient expression of the endocytosis-inducing Nef protein from human immunodeficiency virus or by specific gene silencing mediated by small interfering RNA. In conclusion, this species-adapted procedure for nonviral gene delivery renders primary rodent lymphocytes accessible to rapid functional ex vivo studies, which until now have not been feasible. Furthermore, nucleofection may aid the advancement of therapeutic nonviral gene delivery approaches.
一种能高效地将基因导入原代啮齿动物淋巴细胞的快速方法，将极大地促进对未转化造血细胞中信号传导和代谢途径的研究，以及在敲除或敲低动物模型构建之前对基因表达和靶向策略的验证。在此，我们报告，经过物种适配的核转染程序与优化的培养条件相结合，可使来自广泛使用的大鼠和小鼠品系的增殖原代T细胞、B细胞和自然杀伤细胞易于实现高水平的基因导入。结果，转基因表达水平比既定方案提高了约10至370倍。通过瞬时表达来自人类免疫缺陷病毒的内吞诱导蛋白Nef或通过小干扰RNA介导的特异性基因沉默，使CD4细胞表面分子特异性下调，从而证明了核转染方法用于功能分析的有效性。总之，这种经过物种适配的非病毒基因导入程序，使得原代啮齿动物淋巴细胞能够用于快速的离体功能研究，而这在以前是不可行的。此外，核转染可能有助于推进治疗性非病毒基因导入方法的发展。
