The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.

中文翻译：

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差异RNA干扰：用重组低密度脂蛋白受体相关蛋白（LRP）替代内源性蛋白。

涉及内源基因位点的mRNA表达的干扰常常阻碍了涉及重组cDNA过表达的实验解释。除非可获得来自自然​​发生的突变或敲除小鼠的细胞系，否则需要困难且耗时的基因靶向技术来抑制内源基因表达。使用我们称为“差异RNA干扰”的方法，我们证明了RNA干扰可用于选择性抑制内源基因表达，而不会影响同一蛋白质的共转染重组形式的表达。重组低密度脂蛋白受体相关蛋白（LRP）的功能分析，以研究其参与脂质代谢已被证明是非常困难的，因为它的cDNA较大且缺乏合适的LRP缺陷细胞系。我们构建了一个表达载体，其中包含与EGFP融合的人LRP的全长编码序列，以及表达针对野生型人LRP mRNA（LRP-shRNA）3'-非翻译区的小发夹RNA的载体。当过表达时，标记有EGFP的LRP与内源性LRP共定位并刺激LRP配体的摄取。通过定量RT-PCR，Western印迹和免疫荧光分析判断，LRP-shRNA载体的过表达显着抑制LRP的表达，并显着降低受体相关蛋白（RAP）的摄取。最后，EGFP-LRP和LRP-shRNA载体的共转染证明了内源性LRP表达的选择性抑制，而不影响重组LRP蛋白的同时表达。因此，“差异RNA干扰”的利用提供了一种新的实验方法，以选择性地研究任何给定细胞系中任何重组蛋白的功能而不会干扰内源蛋白表达。