Construction of retrovirol vector containing novel gene mgt -16 and its expression in mouse mesenchymal stem cells

Abstract

Objective To construct the retroviral vector carrying novel gene mgt-16and to investigate its expression in mouse mesenchymal stem cells C3H/10T1/2(10T1/2).Methods DNA sequences containing mouse novel gene mgt-16was used as a template for PCR amplification of full length mgt-16cDNA.Then the DNA fragment was cloned into pEGFP-N1vector to produce pEGFP-N1-16vector after T-A cloning with pMD18Tplasmid and sequencing.The pEGFP-N1-16vector was confirmed by PCR,restriction enzyme digestion and sequencing analysis.The retroviral vector,pLEGFP-N1-16,was constructed using retroviral vector,pLEGFP-N1,and pEGFP-N1-16vector including mgt-16gene.The pLEGFPN1-16vector was verified by restriction enzyme digestion,sequenced,and then transfected into packaging cell line Phoenix to prepare EGFP fused mgt-16retrovirus particles,which were collected and used to infect 10T1/2cells.G418(400μg/mL)continuous selection was conducted to obtain 10T1/2cell clones stably overexpressing EGFP fused mgt-16.Fluorescence microscope was employed to determine the expression and subcellular localization of MGT-16in Phoenix and 10T1/2cells.Results A band of about 300bp size was observed by agarose gel electrophoresis after PCR amplification for mgt-16gene,and the result of sequencing showed that the sequence of insert fragment in T-A clones was identical to mgt-16gene reported in Genbank.PCR,restriction enzyme digestion and sequencing revealed that the pEGFP-N1-16plasmid was successfully constructed.Restriction enzyme digestion and sequencing revealed that the pLEGFP-N1-16plasmid was also successfully constructed.Phoenix and 10T1/2cells overexpressing MGT-16showed green fluorescence distribution in the cytoplasmic,especially around the perinuclear area.Conclusion We have successfully constructed a recombinant retroviral vector carrying novel gene,mgt-16,and expressed it in mouse mesenchymal stem cells,which provides a basis for studying the role of novel gene mgt-16in mesenchymal stem cells.