摘要
目的构建特异性小鼠盐诱导基因(Salt induced kinase2,SIK2)的shRNA腺病毒载体,干扰脂肪细胞中SIK2的表达。方法设计3对靶向SIK2的shRNA,插入穿梭质粒pENTR/U6载体,转染3T3-L1脂肪细胞,通过Real-timePCR方法筛选最佳沉默SIK2效果的穿梭质粒,经腺病毒同源重组后,重组小鼠SIK2shRNA腺病毒载体经脂质体转染HEK293A细胞,在细胞内包装获得腺病毒颗粒AdV-SIK2-shRNA,并进行大量扩增和纯化。纯化的腺病毒感染成熟的3T3-L1脂肪细胞,通过Real-time PCR及免疫印迹法检测鼠SIK2在3T3-L1脂肪细胞中的表达水平。结果纯化后的Ad-shRNA-SIK2重组腺病毒载体可特异下调成熟3T3-L1脂肪细胞SIK2 mRNA和SIK2蛋白的表达。结论干扰3T3-L1脂肪细胞SIK2表达的短发夹RNA重组腺病毒载体构建成功。
Objective To construct the recombinant adenovirus vector of short hairpin interfering RNA（shRNA） against SIK2（Salt induced kinase2） and explore the knocking-down expression of SIK2 in adipocytes.Methods Three short hairpin oligonucleotides and complementary strands were designed to target the consensus sequences of mouse SIK2.The BLOCK-iT RNAi system was used to construct shRNA.Real-time PCR was employed for screening the most effective pENTR/U6-shRNA-SIK2 entry vector.The entry vector was transfected into HEK293A cells via liposome mediation to package and amplify pAd-shRNA-SIK2 adenovirus.3T3-L1 was infected by purified adenovirus,then mouse SIK2 mRNA and protein in 3T3-L1 was detected by Real-time PCR and Western blotting.Results The highly inhibition of mouse SIK2 mRNA and protein were both observed after transfection of 3T3-L1 with adenovirus particles.Conclusion The recombinant adenovirus pAd-shRNA-SIK2 was successfully constructed to knockdown the expression of SIK2 in adipocytes.The cell model of down-expression of the mouse SIK2 was established.This result lays the foundation for further study to investigate the function of SIK2 on obesity.
出处
《中国实验诊断学》
北大核心
2011年第10期1614-1617,共4页
Chinese Journal of Laboratory Diagnosis
基金
深圳市科技计划项目(201002069)
留学回国人员科研启动基金(2009)
