[Construction of short hairpin RNA vector of inhibiting poly ADP-ribose polymerase activity]

To construct expressing vector of short hairpin RNA (shRNA) in order to inhibit human PARP1 activity.

2 pairs of 64 base oligos for hairpin RNA expression which targeted PARP1 gene were chemically synthesized and annealed then ligased with pSIREN-RetroQ vector with BamH II and EcoR I . Cut by EcoR t and Bgl II, shRNA and its upstream U6, which have 330 bp, were inserted into the same treated pEGFP-C1 vecter to construct GFP expression plasmids that inhibited hPARP1 protein shRNA plasmid (pEGFP-C1P). Oligos with a scrambled sequence were used as a negative control.

Recombinant pEGFP-C1P1, pEGFP-C1P2 and pEGFP-C1N vectors was identified by digestion with EcoR I and Bgl II and confirmed by sequencing analysis with U6 primer. The results demonstrated that the 330 bp had been inserted into the expected site. Furthermore, the insertion sequence was exactly correct.

pEGFP-C1-shRNA system has been constructed successfully. This will facilitate the study of PARP1's DNA repairing function.