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Construction of mouse PMX-IRES-Wnt5a retroviral expression vector and expression of Wnt-5a in NIH3T3 cells

Jiong Chen

Abstract

Objective To construct the retroviral expression vector of PMX-IRES-Wnt5a,transfect into 3T3 cells,and observe the expression of Wnt-5a.Methods Gene recombinant technology was employed to clone mouse Wnt-5a gene to the retroviral expression vector of PMX-IRES-GFP,to obtain the recombined plasmid PMX-IRES-Wnt5a,transfect into 3T3 cells by Lipofectamine 2000.The result of transfection was analyzed by RT-PCR and Western-Blotting.Results Identification by enzyme digestion confirmed successful construction of the retroviral expression vector of PMX-IRES-Wnt5a.RT-PCR revealed that the expression of Wnt-5a mRNA and protein was obviously increased in 3T3 cells after transfection.Conclusion The retroviral expression vector of PMX-IRES-Wnt5a is successfully constructed and stably expressed in 3T3 cells.

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Key concepts: Transfection, Lipofectamine, Molecular biology, Viral vector, Expression vector, Vector (molecular biology), Recombinant DNA, clone (Java method)