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目的:构建大鼠Id2基因真核荧光表达载体,并观察外源性Id2基因C_2C_(12)细胞中的表达。方法:RT-PCR扩增出Id2全长cDNA,T4 DNA连接酶将载体pGEM-T和Id2 cDNA进行连接,构建克隆载体,经限制性内切酶EcoR I酶切pGEM-Id2克隆载体和pEGFP-C2真核表达栽体,构建出重组真核表达载体pEGFP-C2-Id2,经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性;通过电穿孔转染法将外源性Id2基因导入C_2C_(12)成肌细胞中。分别于转染4、8、12、24、36、72 h后通过荧光倒置显微镜下观察细胞整体情况,并计算转染效率。结果:经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2 cDNA片段,获得高转染率和高表达外源性Id2基因的C_2C_(12)细胞,转染8 h时,转染效率约为(10.5±2.8)%;转染12 h后,转染效率约为(20.9±3.1)%;转染24 h后,转染效率最高,约为(60.8±3.2)%。结论:成功构建了同时携带有G418筛选位点和Id2基因的真核表达载体;并获得高表达外源性Id2基因的C_2C_(12)细胞。
Objective: To construct eukaryotic expression vector of rat Id2 gene and observe the expression of exogenous Id2 gene in C_2C_ (12) cells. Methods: The full-length cDNA of Id2 was amplified by RT-PCR. The vector pGEM-T and Id2 cDNA were ligated with T4 DNA ligase to construct the cloning vector. The pGEM-Id2 cloning vector and the pEGFP- C2 eukaryotic expression vector to construct a recombinant eukaryotic expression vector pEGFP-C2-Id2, confirmed by enzyme digestion analysis, PCR identification and DNA sequencing correct size of the cDNA fragments and sequences; by electroporation transfected exogenous Id2 gene into C_2C_ (12) myoblasts. The cells were observed under inverted fluorescence microscope at 4,8,12,24,36,72 h after transfection respectively, and the transfection efficiency was calculated. Results: The positive Id2 cDNA fragment of pEGFP-C2-Id2 was confirmed by restriction enzyme digestion and sequence analysis. C 2 C 12 cells with high transfection efficiency and high expression of Id2 gene were obtained and transfected for 8 h , And the transfection efficiency was about (10.5 ± 2.8)%. After transfection for 12 h, the transfection efficiency was about (20.9 ± 3.1)%. After transfection for 24 h, the transfection efficiency was the highest (60.8 ± 3.2)%, . CONCLUSION: The eukaryotic expression vector carrying both the G418 screening site and the Id2 gene was successfully constructed. The C_2C_ (12) cells highly expressing the exogenous Id2 gene were obtained.