Construction and expression of a recombinant lentivirus expression vector of nuclear respiratory factor 1 gene

AIM:To obtain high expression of gene NRF-1 in rat cardiomyocyte cell line H9c2 (2-1) by examining the package of NRF1 lentiviral particles and the effect of transfection to H9c2 (2-1). METHODS: The plasmid of pLenti6.3-NRF1-IRES2-EGFP and two packaging plasmids (pLP1, pLP2 and pLP/VSVG) were packaged into recombinant lentivirus in 293T cells by lipofectamine 2000. The virus was collected and the virus titer was measured. Rat cardiomyocyte cell line H9c2(2-1) was infected with the recombinant lentivirus and positive cells were screened with blasticidin antibiotics. The expression of NRF-1 in H9c2(2-1) cell was identified by fluorescence microscope, PCR and QPCR. RESULTS: The titer of recombinant lentivirus was up to 5.5×107 TU/ml and the green fluorescence was detected in transfection positive target cells using fluorescence microscope. PCR showed that the sequence of recombinant lentivirus was inserted into the target cell genome and QPCR results demonstrated that the expression levels of NRF-1 in positive target cells were 2.25 times higher than those in negative H9c2 cells. CONCLUSION: In this study, expression of gene NRF1 in rat cardiomyocyte H9c2(2-1) has been successfully upregulated, which provides experimental support for further research on myocardial energy metabolism and even the basis for gene therapy of heart failure energy metabolism.