Establishment Of Perilipin Gene Adipose Tissue Specific Expression For Microinjection

Objective:In order to constructe the perilipin transgenic mouse and investigate the function ofperilipin, in the present study we attempted to constructe the vector of transgenic mice modelwhich the perilipin was specific overexpression in adipose tissue. The perilipin gene fragmentfor microinjection was procured .Methods:Firstly, by long PCR, the 6.1kb ap2(fatty acid binding protein)gene fragment whichincludes 5.4kb ap2 promoter was amplified,then it was cloned into pMDTM18-T vector. the5.4kb ap2-promoter was excised with Kpn I and Spe I,then it was exactly confirmed with DNAsequencing.The recombinant vector pMDTM18-T/ap2-promoter was obtained.Secondly, theextraction method of total RNA from adipose tissue was modified to extract high quality totalRNA from C57BL/6J mouse epididymal adipose tissue. By RT-PCR, the full-length perilipincDNA sequence was amplified. Then the full-length perilipin cDNA was cloned into pMDTM18-Tvector. The recombinant vector pMDTM18-T/Plin was confirmed by restriction endonuclease (SpeI and Not I) and DNA sequencing. thirdly, the 5.4kb ap2-promoter fragment from thepMDTM18-T/ap2-promoter was inserted into upper reaches of the recombinant vector pMDTM18-T/Plin in the correct order, to form the recombinant vector pMDTM18-T /ap2-promoter/Plin.Finally,The objective perilipin gene was amplified by restriction digestion of recombinateplasmid pMDTM18-T/ap2-promoter/Plin.The objective perilipin fragment of adipose tissuespecific overexpression was recovered by comparison with the staining gel.It can be used formicroinjectionResults:The recombinant vector pMDTM18-T/ap2-promoter,pMDTM18-T/Plin and pMDTM18-T/ ap2-promoter/Plin were obtained.The recombinant vectors were exactly confirmed by DNA elec-t rophoresis after enzyme restriction and DNA sequencing.The objective perilipin fragmentof adipose tissue specific overexpression was recovered by the staining gel.Its concentr-ation and purity fit in with microinjection.ConclusiConclusions:The transgenic vector pMDTM18-T / ap2-promoter/Plin with perilipin specially expressed inadipose tissues was successfully constructed. The perilipin gene fragment for microinjection wasrecovered. This work has laid foundations for the construction of perilipin transgenic animalmodels.