Identification of ZFP809 sub-domains required for the PBS-dependent gene silencing of MLV-LTR driven transgene expression.

<p>(A) The structures of luciferase reporter vectors used are shown. Black and grey boxes at downstream of the CMV promoter represent the MLV- and dl587rev-derived PBS, respectively. The five nucleotide positions whose base sequence is different between two PBSs are underlined. 293FT cells were co-transfected with one of the reporter vectors and one of the pCMV/flag-X vectors (X denotes either of ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s001" target="_blank">S1B Fig</a>). RRR (relative response ratio) represents the value of the firefly luciferase activity relative to that of the <i>Renilla</i> luciferase activity (control) at 24 hours after transfection. Error bars show the standard deviation obtained from four independent experiments. Asterisks indicate statistical significance (<i>P</i> <0.05) with <i>Bonferroni</i> correction. (B) The structures of the retroviral GFP reporter vectors. When the RNA genome derived from retroviral vectors is reverse transcribed into an RNA-DNA duplex and then into double-stranded DNA, a part of 5' LTR is recombined to a part of 3' LTR. (C) 293FT cells transduced with one of the retroviral vectors (MLV/EGFP or MSCV/EGFP) were sorted based on EGFP expression, and transduced with one of the pLVSIN/CMV/flag-X/IRES/mCherry vectors or the control vector (pLVSIN/CMV/flag/IRES/mCherry). Then, the expression levels of EGFP and mCherry were analyzed at day 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s004" target="_blank">S4 Fig</a>) and day 15 (Fig 3C) after transduction.