Figure 3

Reporter maps. (A) Each reporter contained GFP fused upstream to one of the accessory genes (for shRNAs #0, #1, #6, #7, and #8), a fragment of the core genes (#2, #3, and #9) or a small shRNA-specific target domain (#4 and #5) with stop codons placed between the two domains. Thus, each reporter produced a fused mRNA target composed of GFP plus the HIV-1 sequence from which only the GFP domain was translated. This was engineered to remove the possibility of HIV-1 protein products affecting shRNA activity. (B) We made an all-in-one reporter (the aio sense reporter) to measure the combined activity of simultaneously expressed shRNAs. It had a ~ 400 bp target domain composed of 8 sections of ~ 40 bp covering each ~ 20 bp shRNA target site plus ~ 10 bp either side, and one slightly longer shared section for the two LTR targets since they overlapped each other. Two more reporters were also made (though not shown schematically): the reverse complement of the aio sense reporter (the aio anti-sense) and a non-matched control reporter composed of 7 similarly sized target domains that were unmatched to the chosen shRNAs.