Efficient nonviral gene delivery into primary lymphocytes from rats and mice

被引:50
作者
Goffinet, C [1 ]
Keppler, OT [1 ]
机构
[1] Univ Heidelberg, Dept Virol, D-69120 Heidelberg, Germany
关键词
T cells; B cells; NK cells; nucleofection; rodents;
D O I
10.1096/fj.05-4651fje
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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.
引用
收藏
页码:500 / +
页数:19
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