Construction Of PEGFP-C2-GATA-4 Expression Vector And Expression In P19 Cell

Objective: Transplanting stem cell of different resources to rebuild infarctus cardiac muscle and improving heart function are becoming the focus of curing heart attack. Non-cardiogenic stem cells, such as embryonic stem cell, hemotopoietic stem cell and satellite cell of skeletal muscle, were successfully transplanted into the infarctus cardiac muscle and were observed differentiating into parallel cardiac cells, but the specific mechanism of the transversal differentiation was little known. The development of transplantation and the investigation of the factors and mechanisms about regulating ES cells into cardiac cells suggest that transplantation of ES cells in myocardial infarction has some rationales and good perspect. The further research needs to be done is that what factors and mechanisms are involved in differentiation and regulation of ES cells into cardiac cells.GATA-4 are the important transcription factor in the development process of embryonic heart which is involved in differentiation of cardic precursor cell, cyclization of cordis, septation of compartment, normally functional maintenance of conducting system and mature heart.GATA-4 plays an important role in the development of embryo by its unique zinc fingers. Deletion or mutation of GATA-4 will induce some heart diseases. GATA-4 was one of the earliest biomarker in cardiac precursor cells and necessory to the development of heart. GATA-4 is first expressed in the mesoblast during cardiac protophase and subsequently found in endocardium and cardiac muscle. GATA-4 also regulates development and differentiation of cardiac cell and cardiac tube from cardiac precursor cell.To clone human cardiac muscle-restricted transcription factor GATA-4 cDNA, and construct GATA-4 eukaryotic expression vector, and observe its expression in P19 cell, which will be as the technical base of studying the applied possibility of cardiac muscle-restricted transcription factor GATA-4 in cardiac muscle cell differentiated from embryonic stem cell.Methods: 1 The mouse GATA-4 gene fragment was amplified by PCR from eukaryotic expression plasmid pEFSA-GATA4 and subcloned into pEGFP–C2 vector. Recombinant clones were identified by Kanamycin,and then by bacteria colonies PCR,enzyme analysis and DNA sequencing. 2 The recombinant plasmid was transfected into P19 cells by means of lipoinfection.The expression of GATA4- EGFP fusion protein was comfirmed by immunocytochemistry, Laser Scanning Conforcal Microscope,PCR and Western blot analysis.Results: 1 A fragment about 1325bp was obtained through PCR and proved to be GATA-4 gene cDNA according to its sequence, and further filtered the expression vector pEGFP-C2-GATA-4 by the cutting of restricted enzyme and electrophoresis. 2 Immunocytochemistry, RT-PCR and Western blot were used to demonstrate that GATA-4 was expressed in the transfected P19 cell.Conclusions: We got the fragment of GATA-4 gene and constructed eukaryotic vector pEGFP-C2-GATA-4. In the transfected P19 cell, we found the expression of GATA-4 mRNA. The successfμl conduction of eukaryotic expression vector of GATA-4 cDNA and GATA-4 expression in P19 cell show a great potential value of application. Transcriptional control of cardiac development is estremely complicated. It is a cooperated network system and many transcriptional control factors are involved in which GATA-4 plays a key role. For, GATA-4 regμlates expression not only of cardiac structional genes but also other transcriptional regμlatory factor. Starting from GATA-4, we can draw a complicated transcriptional regulatory network of cardiac development. So we can promote the knowledge of regulatory mechanism of cardiac development and pathogenesis of heart disease, such as CHD. All these we shall do will make the genetic therapy possible in the early stage of heart disease and provide a effective method to radical cure it.