Selection of E. coli strains for stable transformation with recombinant plasmids containing full-length genome of clinical HIV-1 isolates

Strain χ6007 obtained from the parent E. coli strain χ5097 is a result of ptsH5 mutation, which allowed cells to grow without common components of the phosphoenolpyruvate-dependent phosphotransferase system. Segregants of strain χ6007 retaining the Pol+ gene responsible for inability to grow at 37°C, but gaining rifampicin resistance (RifR) were used for cloning of cointegrate plasmids. Preintegration complexes of HIV-1 were cointergated with the pBR-322 plasmid and transformed strain χ6018. Sequencing showed that the pPIC91 hybrid plasmid contains full-length genome of HIV-1 with shortened 5-terminal LTR and full-length copy of pBR322. Elimination of the pPIC91 plasmid from χ6018 cells was followed by the appearance of auxotrophic insertion mutants. Sequencing of the insert region showed that chromosome DNA of the host cell includes integrated genomes of pBR-322 and HIV-1.