Cloning of differentially expressed genes in an HIV-1 resistant T cell clone by rapid subtraction hybridization, RaSH

An HIV-1 resistant T cell clone Rlc2 has been generated that carries mutant , latent HIV-I in a minority of the cell population. Resistant cells expres s HIV-1 receptors CD4 and CXCR4 and display resistance to infection by wild type (wt) HIV-1 at the level of virus transcription. To begin to define th e repertoire of genes modulated in Rlc2 cells that correlate with and poten tially control expression of the HIV-1 resistance phenotype we have employe d a rapid subtraction hybridization (RaSH) technique. For this approach, cD NA Libraries were prepared from double-stranded cDNAs that were enzymatical ly digested into small fragments, ligated to adapters, PCR amplified follow ed by incubation of tester and driver PCR fragments. The RaSH scheme result ed in the cloning of genes displaying differential expression between HIV-1 resistant (Rlc2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of ex pression of the differentially expressed genes documented eleven genes with enhanced (HR clones) and six genes with reduced (HS clones) expression in HIV-1 resistant versus HIV-1 susceptible T-cell clones. (C) 2001 Published by Elsevier Science B.V. All rights reserved.