Objective:To construct the eukaryotic expression vector of pCDNA3.1A-GATA4 and detect its expression in P19 cells. Methods:Because the GC level of the coding sequence of mouse GATA4 was too high, a two step PCR was adopted to clone the full-length coding regions of GATA4,and then the right PCR product was inserted into PMD19-T vector. After DNA sequence analysis,GATA4 was subcloned into pCDNA3.1A vector. Finally,a eukaryotic expression vector of pCDNA 3.1A-GATA4 was obtained. The pCDNA3.1A-GATA4 expression vector was transfected into P19 cells by liposome mediation and the expression was determined by Western blot. Results:The coding sequence of mouse GATA4 gene was successfully amplified and the analysis of DNA sequence approved that the recombinant eukaryotic expression vector contained GATA4 cDNA. Western blot showed that P19 cells transfected with pCDNA3.1A-GATA4 could overexpress the mouse GATA4. Conclusion:The pCDNA3.1A-GATA4 expression vector was constructed successfully and the transfected P19 cells could overexpress mouse GATA4 gene,which pave the way for further studies on the function of GATA4 in the differentiation myocardium and development of heart.