A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain.
A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain.
复制标题
DOI：
10.1093/nar/gkm152
复制
发表时间：
2007
影响因子：
14.9
通讯作者：
Demeneix BA
中科院分区：
文献类型：
--
作者：
Hassani Z;François JC;Alfama G;Dubois GM;Paris M;Giovannangeli C;Demeneix BA
体内特定组织中RNA干扰驱动的功能丧失应允许在时间和空间限定的背景下分析基因功能。然而，在体内将有效的短发夹RNA（shRNA）递送至靶组织仍然存在问题。在这里，我们证明了聚乙烯亚胺（PEI）-交付的shRNA的效率取决于所使用的调控序列，在体内和体外。当在体内进行测试时，由位于H1启动子上游的CMV增强子/启动子组成的杂合构建体产生的shRNA对荧光素酶靶基因的沉默（50%）超过了单独使用H1启动子获得的沉默（20%）。相反，在NIH 3 T3细胞中，H1启动子比杂合构建体更有效（分别抑制75%和60%的靶基因表达）。为了测试CMV-H1 shRNA对体内内源性基因的效率，我们使用了针对甲状腺激素受体α1（TRα1）的shRNA。当在小鼠脑中载体化时，杂交构建体强烈地去阻遏CyclinD 1-荧光素酶报告基因表达，CyclinD 1是负调控的甲状腺激素靶基因。我们得出结论，启动子的选择影响shRNA的效率明显在不同的体外和体内的情况下，混合CMV-H1构建体是最佳的shRNA在小鼠大脑中的交付。
RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor α1 (TRα1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain.