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212 Zhang et al.Going one step beyond simple knockdown of gene expression, one can useRNAi technology to knockdown expression of an endogenous protein andsimultaneously replace it with a mutant version of the same protein. This isanalogous to a gene “knockin” experiment, and has great potential for definingfunctional domains of proteins using physiological assays, without interferencefrom the wild-type endogenous protein. Herein, a single retrovirus has beendescribed that encodes a RNAi to knockdown the endogenous protein and aRNAi-resistant mRNA that, in turn, codes for a wild-type or mutant version ofthe same protein. The mRNA is made RNAi-resistant by introducing silentmutations into the redundant positions of each codon, so that they do not affectthe amino acid sequence. The virus is designed so that the individual shRNAand cDNA-expression cassettes corresponding to any gene of interest can bereadily subcloned into the virus.In this chapter, it is assumed that the reader has identified a gene of interestand the cDNA(s) encoding the mutant or mutants of interest. First, how to generatea functional shRNA that knocks down the target protein is described. Forthis purpose, the polymerase chain reaction (PCR)-shagging approach ofHannon and coworkers is used (7). However, an entry vector for the shRNA,called pPUR V2 has been custom designed. Once the shRNA-expression cassetteis subcloned into this vector, it can be readily transfected into a transfectablehuman cell line, the transfected cells selected in puromycin can then beassayed for knockdown of the target protein. Functional shRNAs are then easilysubcloned from pPUR V2 into the retrovirus plasmid. The retrovirus is modifiedfrom the pQCXI series of vectors (Clontech: Mountain View, CA). The vectorhas been designed with the capacity to direct expression of the shRNAsubcloned from pPUR V2, a cDNA coding for a shRNA-resistant mRNA, anda gene-encoding resistance to puromycin. The retrovirus plasmid is packagedinto infectious retrovirus using Phoenix cells and then delivered to the targetcells by a standard virus infection.2. Materials1. pPUR V2—this cloning vector was modified from pPUR (Clontech). An approx750-bp BamHI/EcoRI fragment was excised from pPUR and replaced by asynthetic oligonucleotide linker containing the multiple cloning site (MCS) inFig. 1. The linker was synthesized with BglII and MfeI sticky ends, which arecompatible with BamHI and EcoRI, respectively, but meaning that the originalBamHI and EcoRI sites are destroyed by the ligation. This plasmid is availableon request.2. pGEM1-U6 plasmid (a gift of Greg Hannon [7]). This plasmid contains the humanU6 promoter, a promoter that directs expression by RNA polymerase III.3. 5 U/µL Taq polymerase (Invitrogen).