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1. Select for latent infection. 2. Map integration sites. 3. siRNA library in individual clones to ID human regions which affect activation. HIV-1. Massively Parallel Pyro - sequencing. Jurkat Cells +HAART. HAART kills active HIV populations, selecting for latent infections.
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1. Select for latent infection 2. Map integration sites 3. siRNA library in individual clones to ID human regions which affect activation HIV-1 Massively Parallel Pyro- sequencing Jurkat Cells +HAART HAART kills active HIV populations, selecting for latent infections + or - Prostratin and valproic acid 4. Larger Scale low throughput verification 5. Check for survival due to integration site bias Pool Massively Parallel Pyro- sequencing siRNAs affecting latent HIV activation Bioinformatics Figure 2. Technique overview
3. Select for latently infected populations by treatment with HAART 1. Infect Jurkat cells with HIV 2. Verify Infection and HAART sensitivity HIV-1 * GFP Infected Cells +HAART Jurkat Cells Infected Cells • Verify GFP expression (ie active • infection) in 90%+ of clones by FACS • Test a sample pool for sensitivity to • various drug cocktails 5. Repeat infection until most cells are latently infected 4. Verify Latent Integration Latently Infected Actively Infected Uninfected HIV Infected Cells +HAART actin Allow cells to grow and then qPCR a sample with primers against viral genome to estimate percent latently infected versus uninfected cells Figure 1. Selection of latently infected cells
Table 1. siRNA screens to identify RNAs involved in latent HIV activation