Optimization of ADIPOQ shRNA transfection system in porcine preadipocyte

Four pairs of specific ADIPOQ shRNA oligoes were designed and synthesized,according to ADIPOQ cDNA sequence and the principle of shRNA designing.The complement form was obtained by annealing and cloned into plasmid vector pGPU6/GFP/Neo,then the recombinant plasmid was transformed into strain DH5α.Finally the vectors were identified by restriction enzyme analysis and DNA sequencing.The eukaryotie expression vectors were transfected into preadipocytes.The recombinant plasmid was cloned,and the sequence was obtained.In the preadipocytes transfected with the recombinant vectors,the expression of green fluorescent protein(GFP) was detected.The optimal ratio of plasmids and lipofectamine 2000 is 0.6 μg,lipofectamine 2000 is 1.2 μL,quality/volume ratio of plasmid concentration and lipofectamine 2000 is 1∶2,the transfection efficiency is the highest up to 53.9%.Pig ADIPOQ shRNA eukaryotic expression vector was constructed and identified successfully,then was transfected into preadipocytes successfully.Finally,the optimal ratio of plasmids and lipofectamine 2000 was determined according to the highest transfection efficiency,which established a favorable foundation for further study on the function of ADIPOQ.