A rapid and simple method for the isolation of mutant variants regulating tissue-specific expression of the TnI gene through drug selection

Abstract

TnINEO fusion gene was constructed by fusing 3.4-kbp of quail TnI genomic DNA sequences spanning the promoter to exon 5 and a neo gene in frame. A myoblast cell line was established after transfection of pTnINEO. Since this cell line was passaged several times, a high frequency of neomycin (G418) sensitivity conversion was detected. Two drug-resistant variants were analyzed through genomic Southern blot and S1 nuclease protection assay. One variant has a mutation(s) in the regulatory element that activated the dormant TnI promoter-enhancer in myoblast, and the other has shown the genomic rearrangement. This result presented the possibility of isolating factor(s) that activate the muscle-specific TnI promoter simply by screening drug-resistant cells having appropriate mutations.