Construction Of Eukaryotic Expression Vector Containing Human Cardiac α-actin Promoter And It's Application In ES Cells

The death of highly vulnerable cardiomyocytes leads to cardiac dysfunction, includingheart failure,myocardium injury and infarction. Because cardiomyocyte is end-stagedifferentiating cell and cann't regenerate, the quest for suitable alternative donor cells hasbeen a hot research field. Embryonic stem cells(ES cells),present in the early stages ofembryo development, are capable of unlimited, undifferentiated proliferation in vitro.Totipotent ES cells can differentiate into a wide variety of cell types in defined medium andenvironment that make them attractive candidates for use in a series of innovativetransplantation paradigms, especially transplantation of cardiomyocytes. Exploitation of EScell-derived cardiomyocytes may facilitate the analysis of early cardiac development, andpave the way for novel therapeutic modalities for enhancing myocardial performance andtreating cardiac disease. There are some difficulties in Inducing ES cells into speciallycardiomyocytes, its essential to label and sort the pure differentiate cardiomyocytes at stagesof differentiation. Cardiacα-actin is one of the earliest markers of cardiac cell development which isexpressed in the early developing heart and observed in all somites as they formed. Cardiacα-actin promoter has been shown to be cardiac specific and switched on before the onset ofspontaneous contractions in ES cells and be sufficient to direct cardiac-specific geneexpression in stable transformants, so we human cardiac α-actin promoter for the genetargeting in this study. Human cardiac α-actin promoter obtained from the human heartgenome by polymerse chain reaction(PCR) was cloned into pMD-18T vector,and was namedpMD-18T-α-actin-P. The EcoRI/HindIII fragment of pMD-18T-α-actin-P was ligated intothe same fragment of pEGFP-N1(４.1kb,moved CMV promoter by AaeI/BagII).Theconstructed expression vector, namedα-actin-pEGFP-N1 which contains neor gene and GFPgene as positive selection markers,was transferred into mice ES cells by Lipofectamine2000 This study was divided two parts:1. According to the published DNA sequence of humancardiac α -actin promoter,we designed one pair of primer,which contains specificenzyme-degesting locus at the 5' of each primer to guarantee the orient ligation in the vectorand using PCR method we cloned the complete DNA sequence of it. The analysis of thesequencing result shows that it is a member of the human gene family and also shares 72%amino acid sequence identity with standard human cardiac α-actin promoter. Analysis of thepromoter sequence contains all regulatory factor of the standard promoter and has similarlytransmembrane region; 2. The recombinant constructureα-actin-pEGFP-N1was finished,using purified EGFP to transfect mice ES cells in different condition, it's found that (1) DNAconcentration should be 3.0~5.0μg/ml, transfer time was 2~3h; (2) In this experiment ,it wasselected for 7days in G418 to kill the nontransfected cell and 200μg/mL G418 was used asperfect working concentration; (3) The results of fluorescent observation andimmunocytochemistry assay showed that the 454bp promoter sequence was expressed andhas strong regulative capacity.