Construction of fusion expression vector EGFP-PDX-1 and its transfection into rat fetal hepatic stem cells by electroporation / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct the fusion expression vector of pancreatic-duodenal homeobox gene 1 (PDX-1) fused to green fluorescent protein (GFP) capable of stable expression in fetal rat hepatic stem cells after transfection by electroporation.</p><p><b>METHODS</b>PDX-1 cDNA was amplified from SK900/BLSCRIPT plasmid and cloned into the multiple cloning site of pEGFP-C1 to obtain the recombined plasmid pEGFP-C1-PDX-1. Rat fetal hepatic stem cells were isolated, cultured, identified and transfected with the recombinant vector by electroporation, followed by observation of these cells with fluorescent microscope. The result of transfection was analyzed by RT-PCR and cell growth curve.</p><p><b>RESULTS</b>Identification by enzyme digestion confirmed successful construction of the recombinant vector. Fetal hepatic stem cells can stably express GFP and PDX-1 for a period of time, and their growth and proliferation was not obviously affected after transfection.</p><p><b>CONCLUSION</b>The fusion expression vector of EGFP-PDX-1 is successfully constructed and stably expressed in rat fetal hepatic stem cells, which may facilitate the study of the role of PDX-1 in stem cell differentiation into insulin-producing cells.</p>