Construction of mammalian expressing vector pEGFP-PDX-1

Objective: To study the induction mechanism of PDX-1,the key gene to regulate the development of pancreatic endocrine cells,during the differentiation of hepatic stem cells into insulin-producing cells,we try to construct a mammalian-expressing vector carrying PDX-1 to search for a proper way to induce the differentiation of hepatic stem cells into islet β-cells from genic level.Methods: All experiment methods are performed according to the standard protocols of molecular clone technology.Polymerase chain reaction（PCR） was used to proliferate PDX-1 cDNA from SK900/BLSCRIPT plasmid,producing an 858bp band with HindⅢ and BamHⅠat both sides of the DNA.Cloned PDX-1 cDNA into the MCS of pEGFP-C1 to get a recombined mammalian expressing plasmid pEGFP-C1-PDX-1,then SmaⅠdigested and separated by 0.8% agrose gel to identify the recombined plasmid.Results: We constructed successfully a mammalian-expressing vector pEGFP-PDX-1.The recombined plasmid has the same open reading frame as pEGFP and perfect potential to translate and express in the mammalian cells.Conclusion: PDX1 was cloned into the C terminus of pEGFP-C1.Under the control of enhancer and promotor of pEGFP-C1,PDX-1 would be（expressed） as fusions with pEGFP-C1, increasing the translation efficiency of PDX-1 in mammalian cells without changing the structure and function of its translating protein.In addition,the recombined vector chooses GFP as a report gene to permit the positive cells to be selected in a simple way from the primary cell level.PDX-1;gene expression;pEGFP;PCR