Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells
- VernacularTitle:小鼠pdx-1基因真核表达载体的构建及其在胚胎干细胞中的表达
- Author:
Guangji ZHOU
;
Haiwei XU
;
Li YANG
;
Jun TANG
;
Dabing LI
;
Jifu QU
- Publication Type:Journal Article
- Keywords:
Genes, pdx-1;
Embryonic stem cells;
Eukaryotic expression vector
- From:
Chinese Journal of Pathophysiology
1989;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5? was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5? was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.
