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218 Zhang et al.Fig. 4. Schematic map of modified pQCXIP plasmid. This retrovirus plasmid isdesigned for simultaneous expression of both shASF1a, an shASF1a-resistant wildtypeor mutant ASF1a cDNA, and resistance to puromycin under control of the indicatedpromoters. See text for details.This vector was made as follows. First a puromycin-resistance gene wasinserted into the MCS of pQCXIN as a BamHI/EcoRI fragment. This fragmentwas generated by PCR using pQCXIP as a template and PCR primers containinga BamHI site (forward primer, 5′-end of gene) and an EcoRI site (reverseprimer, 3′-end of gene). Second, using a PCR-based approach, the neomycinresistancegene downstream of the internal ribosomal entry site in pQCXIN wasremoved and replaced with a shRNA-resistant ASF1a cDNA flanked by a uniqueXhoI site at the 5′-end and a unique MluI site at the 3′-end. The cDNA codes foran hemagglutinin (HA)-tagged form of ASF1a. This cDNA can be excised withXhoI and MluI and replaced by another cDNA of choice. Third, the U6-shASF1acassette was excised from pPUR V2 as a NheI fragment and inserted into theunique NheI site in the 3′-LTR of the virus plasmid. This vector was verified bysequencing and is available on request.