The human immunodeficiency virus (HIV-1) infects and kills CD4+ T-lymphocytes causing a progressive loss of host immune competence, which ultimately leads to AIDS. RNA interference, as mediated by short interfering RNAs (siRNAs) designed to target viral mRNAs and expressed endogenously, offer a potential gene therapy approach to inhibit HIV replication. However, a simple and reliable method to screen the silencing activity of particular anti-HIV siRNAs is useful prior to conducting more extensive experimentation to determine the downstream effects on viral replication. Here, a short hairpin RNA (shRNA) targeting HIV-1 Rev (Revsh8526) was designed and cloned into an expression plasmid under the control of the RNA Polymerase III H1 promoter. To test the ability of Revsh8526 to silence Rev, a chimeric β-galactosidase reporter plasmid containing Rev exon 2 (β-galactosidase-RevE2) was generated. The ability of Revsh8526 to target and inhibit reporter gene expression was shown by X-gal staining and ortho-nitrophenyl-β-galactoside (ONPG) assay. The results of these assays indicated Revsh8526 effectively targeted and significantly inhibited β-galactosidase expression as compared to a non-targeted shRNA. These results show the utility of the chimeric β-galactosidase reporter in initial screening of siRNA reagents.
Tager, Kylie R. and Jackson, William H.
"A Simple and Reliable Reporter Assay to Test siRNA-Mediated Silencing of HIV-1 Gene Functions,"
Journal of the South Carolina Academy of Science: Vol. 19:
2, Article 5.
Available at: https://scholarcommons.sc.edu/jscas/vol19/iss2/5