A system for Cre-regulated RNA interference in vivo
A system for Cre-regulated RNA interference in vivo
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DOI：
10.1073/pnas.0806907105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Hynes，Richard O.
中科院分区：
文献类型：
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作者：
Stern，Patrick;Astrof，Sophie;Hynes，Richard O.
我们报告了一个Cre调控的RNA干扰素在体内表达的系统。表达盒包含可选择的和FACS可分选的标记，其与另外的标记基因和反义方向的shRNA串联。这些盒的两侧是串联的LoxP位点，排列方式使Cre表达逆转标记物-shRNA构建体，允许其调节表达（同时删除原始选择/标记基因）。所述盒可被掺入逆转录病毒或慢病毒载体中并递送至培养中的细胞或用于产生转基因小鼠。我们描述了包含报告基因、基于miRNA的RNAi（包括一次两个shRNA构建体）和癌基因的各种组合的盒，并证明了在培养的细胞中有效的RNA干扰的递送，在其后代中有效转导到具有细胞类型特异性敲低的造血干细胞中，以及在转基因小鼠中快速产生受调控的shRNA敲低。这些载体系统允许在体外和体内多系统中调控基因表达的组合操作（过度表达和功能丧失）。
We report a system for Cre-regulated expression of RNA interferencein 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 systemsin vitroandin vivo.