Construction Of Human PPARγ Lentivirus Overexpressing Vector

BACKGROUND:Peroxisome proliferator-activated receptor(PPAR)is a member of the nuclear receptor transcription factor supernumerine,which regulates the expression of target gene.According to the structure,PPAR can be divided intoα,β(orδ)andγthree types,Which PPARγmainly expressed in adipose tissue and immune system,and adipocyte differentiation,immune and insulin resistance are closely related.In the present study,PPARγis associated with insulin resistance in type 2 diabetes mellitus,and is the target molecule for the action of troglitazone(TZDs),which regulates the sensitivity of cells to insulin and promotes the cell’s sugar metabolism.However,due to individual differences,20%to 30%of patients with type 2 diabetes did not respond to TZD,and TZDs had significantside effects,such as water and sodium retention lead to heart failure,fract-ure and other risksAIMS:In the PPARγ-related pathway to find a new insulin sensitivit-y target to address the individual drug target individual differences,and to further clarify the mechanism of insulin resistance.METHODS:1.The primers were designed according to the gene sequ-ence of PPARγin human cDNA library,the plasmid pGEM-PPAR-γwas used as template to amplify the target gene human PPARγ;2.NheI and NotI were digested at both ends of the primer,and then NheI and NotI were digested with the vector plasmid pCDH-C MV-MCS-EF1-GFP-T2A-Puro and the target gene Human PPARγ;3.TheplasmidpCDH-CMV-MCS-EF1-GFP-T2A-Purorecovery connect a large fragment of human PPARγfragments;4.The transformants were extracted and the plasmids were extractedwith NheI+NotI.The positive clones were identified by restriction enzyme digestion and sequenced.5.The recombinant plasmids containing the target gene were introd-uced into 293T cells to produce lentivirus with high titer containing the target gene,namely LV-hPPARγ-GFP-Puro lentivirus,referred to as LV-hPPARγ.RESULTS:The lentiviral expression vector pCDH-CMV-hPPARγ-EF1-CoGFP-T2A-Puro containing the target gene was successfully constr-ucted.The lentiviral expression of LV-h PPARγwas 1×10~8TU/ml.