Abstract:

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.

Key words:  mouse; PID1 gene; RNA interference; expression vector