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Design and Application of a shRNA 217Fig. 3. Design of an shRNA-resistant ASF1a. The figure shows the region of thewild-type endogenous ASF1a mRNA that is targeted by the shRNA (Line 2); the correspondingamino acid sequence (Line 1); and the ectopically expressed ASF1a mRNAthat does not change the protein sequence, but which is resistant to the shRNA by virtueof base changes in the third position of each codon.plasmid Maxi kit, according to the manufacturer’s instructions. Use each of thesepurified plasmids to transfect a 60% confluent 10-cm plate of U2OS cells by thecalcium phosphate method (9). In addition, transfect one plate with pPUR V2vector alone and one plate with no DNA. Add puromycin to a final concentrationof 1 µg/mL, 48 h after transfection (see Note 1). After 72–96 h, scrape the cells intoLaemmli sample buffer, determine the protein concentration by Bradford assay andfractionate 50–100 µg of total cellular protein by SDS-PAGE (10) (see Note 2).9. Western blotting to test for protein knockdown: transfer the proteins from the gelto a PVDF membrane and immunoblot to detect the protein of interest using astandard protocol (10). The extract derived from pPUR V2-transfected cells servesas a positive control and the efficiency of knockdown is measured relative to this.If antibodies to the protein of interest are not available, knockdown can be assayedby quantitative real-time RT-PCR.3.2. Design of shRNA-Resistant cDNATo design an shRNA-resistant cDNA, silent mutations are introduced in thecDNA in the region targeted by the shRNA (Fig. 3). These mutations changethe nucleotide sequence, but do not affect the encoded protein sequence. Formost codons, this means changing the third base of the codon. In other cases,more or less flexibility is allowed. Consult the full genetic code for details (e.g.,in the New England Biolabs catalog). Mutations are introduced by a standardmutagenesis protocol, for example, two-step mutagenic PCR or with theStrategene Quickchange kit (La Jolla, CA) (8).3.3. Subcloning shRNA and cDNA Into RetrovirusA modified version was constructed of pQCXIN (Clontech, http://orders.clontech.com/clontech/techinfo/vectors/vectorsM-Q/pQCXIN.shtml) (Fig. 4).The final vector encodes the ASF1a shRNA, under control of the U6 promoter;a puromycin-resistance gene, under control of a CMV promoter; and an ASF1acDNA that is resistant to the ASF1a shRNA, under control of the same CMVpromoter and an internal ribosomal entry site.