[Construction and identification of the recombinant adenovirus expressing the short hairpin RNA targeting phosphatase and tensin homolog deleted on chromosome ten gene].

OBJECTIVE

To construct the recombinant adenovirus expression vector of a short hairpin RNA (shRNA) targeting phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene for gene therapy of ischemic cerebral injury.

METHODS

The U6 expression promoter and shRNA of pGenesil-1-shRNA, which was constructed and identified in our previous experiment, were subcloned to pAdTrack shuttle plasmid. The product pAdTrack-U6-shRNA was linearized by PmeI for homologous recombination with pAdEasy-1 in pAdEasy-1 competence bacteria. The positive clone was identified by enzyme digestion, PCR analysis and DNA sequence analysis. After linearization by PacI, the recombinant adenovirus DNA shuttle plasmid pAdEasy-U6-shRNA was transfected into 293 cells for packaging and amplification of Ad-U6-shRNA, which was further identified by PCR analysis and DNA sequence analysis. Western blotting was used to detect the expression of PTEN protein in the hippocampal neurons infected with the adenovirus.

RESULTS

The pAdTrack-U6-shRNA and pAd-U6-shRNA plasmids had been successfully constructed as verified by PCR analysis, enzyme digestion and DNA sequence analysis. PCR analysis and DNA sequence analysis confirmed successful packaging of the recombinant adenovirus Ad-U6-shRNA in 293 cells. PTEN protein expression decreased significantly in the hippocampal neurons after infection by the recombinant virus.

CONCLUSION

We have successfully constructed the recombinant adenovirus Ad-U6-shRNA targeting PTEN gene, which provides a basis for investigating the role of PTEN in neuroprotection after cerebral ischemic injury using RNA interference.