Stern Patrick, Astrof Sophie, Erkeland Stefan J, Schustak Joshua, Sharp Phillip A, Hynes Richard O
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13895-900. doi: 10.1073/pnas.0806907105. Epub 2008 Sep 8.
We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo.
我们报道了一种用于体内Cre调节的RNA干扰表达系统。表达盒包含与其他标记基因串联的可选择和可通过荧光激活细胞分选（FACS）分选的标记，以及反义方向的shRNA。这些表达盒两侧是串联排列的LoxP位点，使得Cre表达能使标记-shRNA构建体发生倒转，从而实现其调节表达（同时删除原始的选择/标记基因）。这些表达盒可整合到逆转录病毒或慢病毒载体中，并递送至培养细胞，或用于生成转基因小鼠。我们描述了包含各种报告基因组合、基于miRNA的RNA干扰（包括同时使用两个shRNA构建体）和癌基因的表达盒，并展示了在培养细胞中有效RNA干扰的递送、在造血干细胞中高效转导并在其后代中实现细胞类型特异性敲低，以及在转基因小鼠中快速产生受调节的shRNA敲低。这些载体系统允许在体外和体内的多个系统中对基因表达进行受调节的组合操作（包括过表达和功能丧失）。
