RNA干扰沉默PID1基因在C2C12细胞中表达的研究

 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，24h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。

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

To construct short hairpin RNA (shRNA) expression vector that can interference PID1 expression in mouse.  Four short hairpin RNA sequences and the negative interfering sequences were designed through mouse PID1 cDNA sequence. Then four corresponding double stranded DNA sequences were used to construct pGPU6/GFP/Neo vector, namely pGPU6/GFP/Neo-PID1-1, pGPU6/GFP/Neo-PID1-2, pGPU6/GFP/Neo-PID1-3, pGPU6/GFP/Neo-PID1-4 and pGPU6/GFP/Neo shRNA NC respectively. They were transfected into the C2C12 cells, in which the silencing or expressing down effect on PID1 expression was investigated by RT PCR and Western blot. The expression vector targeting on PID1 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 PID1 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/Neo-PID1-3 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.