RNA干扰沉默PID1基因在C2C12细胞中表达的研究  

Silence of PID1 gene expression using RNA interference in C2C12 cell line

作　　者：杨伦[1] 徐正刚[1] 王慧[1] 陈其美[1] 陈伟[1] 胡艳霞[1] 石元[1] 祝洪磊[1] 曾勇庆[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《山东大学学报（理学版）》2013年第1期36-42,共7页Journal of Shandong University(Natural Science)

基　　金：国家转基因重大专项资助项目(2011ZX08006-002);国家高技术研究发展计划"863"重点资助项目(2008AA101008);山东省现代农业(生猪)产业技术体系建设专项资助项目;山东省农业良种工程重大资助项目(2011LZ012-03;2011LZ015)

摘　　要：构建和筛选对PID1(phosphotyrosine interaction domain containing 1,PID1)基因有RNA干扰作用的PID1-shRNA表达载体。据小鼠PID1 cDNA序列,优化设计了4条shRNA及1条阴性干扰序列,插入pGPU6/GFP/Neo载体中,得到pGPU6/GFP/Neo-PID1-1、pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3、pGPU6/GFP/Neo-PID1-4和pGPU6/GFP/Neo-PID1-NC。干扰载体转染C2C12细胞,以RT-PCR和Western blot技术检测shRNA对C2C12细胞中PID1 mRNA和蛋白表达的下调作用。结果表明:靶向PID1基因的4个shRNA重组质粒载体经测序分析,其shRNA编码序列与预期设计的完全一致,经酶切鉴定和测序分析证实,靶向PID1基因的shRNA重组质粒载体构建成功。进一步将构建的4个表达载体分别转染C2C12细胞,24 h后细胞中PID1基因mRNA表达水平依次下调(23.58±1.87)%、(75.44±0.77)%、(70.52±0.41)%和(56.60±3.13)%。48 h后细胞中PID1蛋白表达水平依次降低(30.15±5.05)%、(71.86±4.85)%、(67.93±2.28)%和(56.81±2.01)%。所筛选出的pGPU6/GFP/Neo-PID1-2、pGPU6/GFP/Neo-PID1-3和pGPU6/GFP/Neo-PID1-4三个表达载体均能高效地抑制转染细胞PID1 mRNA和蛋白的表达,为进一步研究PID1基因的功能奠定了基础。To construct short hairpin RNA （shRNA） expression vector that can interference PIDI expression in mouse. Four short hairpin RNA sequences and the negative interfering sequences were designed through mouse PIDI cDNA se- quence. Then four corresponding double-stranded DNA sequences were used to construct pGPU6/GFP/Neo vector, namely pGPU6/GFP/Neo-PIDI-1, pGPU6/GFP/Neo-PID1-2, pGPU6/GFP/Neo-PID1-3, pGPU6/GFP/Neo-PIDI4 and pGPU6/GFP/Neo-shRNA-NC respectively. They were transfected into the C2C12 cells, in which the silencing or expressing down effect on P1D1 expression was investigated by RT-PCR and Western blot. The expression vector targe- ting on P1D1 were successfully constructed, and confirmed by sequence analysis. Four expression vectors generated by insert double-stranded DNA sequences, were transfected into the C2C12 cells, after 24 h, the P1DI mRNA expression was decreased by （23.58±1.87）%, （75.44 ±0.77）%, （70.52 ±0.41）% and （56.60 ±3. 13）%, respectively, and after 48 h, its protein expression was decreased by （30.15 ±5.05 ） % , （71.86 ±4.85 ） % , （ 67.93 ±2.28 ） % and （ 56.81 ±2.01 ） % , correspondingly. The constructed expression vector pGPU6/GFP/Neo-PID1-2, pGPU6/GFP/NeoPID1-3 and pGPU6/GFP/Neo-PID1-4 can effectively inhibit the expression of PID1 mRNA and protein, which could provide a basis for further research on the function and mechanism of PID1.

关 键 词：小鼠 PID1基因 RNA干扰 表达载体 

分 类 号：Q953[生物学—动物学]