[Construction of fusion expression vector EGFP-PDX-1 and its transfection into rat fetal hepatic stem cells by electroporation].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Sun
Bing
B
Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China. sunice116@163.com
Sun
Xiao-yan
XY
An
Jing
J
D000818:Animals; D018274:Electroporation; D005260:Female; D053686:Fetal Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods
D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C485184:enhanced green fluorescent protein; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins