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 R1c2 has been generated that carries mutant, latent HIV-1 in a minority of the cell population. Resistant cells express 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 the repertoire of genes modulated in R1c2 cells that correlate with and potentially control expression of the HIV-1 resistance phenotype we have employed a rapid subtraction hybridization (RaSH) technique. For this approach, cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified followed by incubation of tester and driver PCR fragments. The RaSH scheme resulted in the cloning of genes displaying differential expression between HIV-1 resistant (R1c2) and susceptible (SupT1) cells, including known genes and those not described in current DNA databases. Analysis of the pattern of expression 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.
Simm
M
M
Molecular Virology Laboratory, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, NY 10019, USA.
Su
Z
Z
Huang
E Y
EY
Chen
Y
Y
Jiang
H
H
Volsky
D J
DJ
Fisher
P B
PB
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D020869:Gene Expression Profiling; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000821:virology*; D013997:Time Factors