Construction of bromodomain-deleted BRD7 mutation vector based on homologous recombination and reverse PCR amplification / 中南大学学报(医学版)

ABSTRACT

OBJECTIVE@#To construct a eukaryotic expression vector of bromodomain-containing protein 7 (BRD7) with deletion of bromodomain (BRD7△brd) using the homologous recombination and reverse PCR amplification techniques.@*METHODS@#The linear DNA fragments of bromodomain-deleted mutation of BRD7 (pIRES2-EGFP- 3Flag/BRD7△brd) were amplified by one pair of reverse PCR primers using high-fidelity enzyme, and then these fragments were transformed into E.coli to obtain the eukaryotic expression vector expressing BRD7△brd protein based on homologous recombination and plasmid cyclization.@*RESULTS@#Bromodomain-deleted clones were identified by digestion with restrictive enzymes, and then the sequence and protein expression were further confirmed by sequencing and Western blot assays. The results suggest that pIRES2-EGFP-3Flag/BRD7△brd was successfully constructed.@*CONCLUSION@#We establish a simple and quick method to construct plasmids with pIRES2-EGFP- 3Flag/BRD7△brd using reverse PCR amplification and homologous recombination techniques. We also found that the concentration of template in PCR reaction system is one of the critical factors that affect the rate of homologous recombination. Of all, this improved technique could be widely used in the construction of gene mutations.