ADIPOQ shRNA在猪前体脂肪细胞中转染体系的优化  

Optimization of ADIPOQ shRNA transfection system in porcine preadipocyte

作　　者：李付娟[1,2] 王帅[1,2] 朱亚楠[1,2] 任久生[1,2] 柳思源[1,2] 徐靖博[1,2] 张阳阳[1,2] 孙广杰[1,2] 高妍[2] 张嘉保[2] 陈承祯[2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学实验动物中心,吉林长春130062

出　　处：《中国兽医学报》2013年第4期616-621,626,共7页Chinese Journal of Veterinary Science

基　　金：吉林省重大科技专项资助项目(20065019)

摘　　要：根据Genbank提供的猪ADIPOQ基因序列及shRNA设计原则,化学合成4段编码短发夹RNA的寡核苷酸序列,将其定向克隆到pGPU6/GFP/Neo中U6启动子的下游,转化DH5α菌株,双酶切鉴定正确后进行测序,将构建好的真核表达载体转染前体脂肪细胞,并对条件进行优化。限制性内切酶PstⅠ和BamHⅠ酶切显示设计合成的shRNA编码序列被成功插入pGPU6/GFP/Neo质粒中,测序结果显示插入片断与设计序列完全一致,证明重组质粒构建成功,转入到前体脂肪细胞中,可以看见绿色荧光蛋白表达。当质粒浓度0.6μg,脂质体2000是1.2μL,两者质量/体积比为l∶2时,此时转染效率最高,达到53.9%。成功构建了猪ADIPOQ基因的shRNA真核表达载体,并成功将该质粒转入猪的前体脂肪细胞中去,还对质粒与脂质体2000用量比条件进行了优化,此举能为进一步研究ADI-POQ在猪前体脂肪细胞中的功能奠定了良好的基础。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 ob- tained by annealing and cloned into plasmid vector pGPU6/GFP/Neo, then the recombinant plasmid was transformed into strain DHSa. Finally the vectors were identified by restriction enzyme analysis and DNA sequencing. The eukaryotie expression vectors were transfeeted 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.

关 键 词：ADIPOQ基因 SHRNA 真核表达载体 RNAI 前体脂肪细胞 

分 类 号：Q78[生物学—分子生物学]