Figures & data
Fig. 1. Schematic diagram of fluorescent reporter-containing vector system combined with flow cytometry methodology.
Notes: (A) Structures of the retroviral vectors. When the RNA genome derived from retroviral vectors is reverse transcribed into an RNA-DNA duplex and then into double-strand DNA, a part of 5′ LTR is recombined to a part of 3′ LTR.Citation28) (B) The development of the experimental system for monitoring of gene silencing over a longtime period. (C) Interpretation of FACS analysis.
Fig. 2. Long-term monitoring of ZFP809-mediated gene silencing effect on transgene expression driven by MLV.
Notes: (A, B) Ba/F3 and K562 transduced with MLV/EGFP or MSCV/EGFP were sorted based on EGFP expression and transduced with MSCV/ZFP_huKO, followed by the analysis of the expressions of EGFP and huKO on Day 3 and 24 after transduction.(C) EGFP expression was measured by RT-PCR in K562 cells analyzed with FACS (Fig. (B)). β-actin was used for a loading control.
Fig. 3. Long-term monitoring of ZFP809-mediated gene silencing effect on transgene expression driven by CMV or EF1-α promoters.
Notes: (A) Constructs of the CMV vectors expressing EGFP. Shadow box and closed box located downstream of the CMV promoter are the MLV- and dl587rev-derived PBS, respectively. HEK293 was transfected with the pCMV_MLV/I/EGFP or pCMV_dl587/I/EGFP and analyzed on EGFP and huKO expression with FACSAria on Day 3 and 17 after transduction with MSCV/ZFP_huKO. (B) Constructs of the EF1-α vectors expressing EGFP. Shadow box and closed box located downstream of the EF1-α promoter are the MLV- and dl587rev-derived PBS, respectively. HEK293 transfected with the EGFP expression vectors driven by the EF1-α promoter with the MLV- or dl587rev-derived PBS downstream of the promoter, respectively (pEF1_MLV/I/EGFP or pEF1_dl587/I/EGFP). The cells were transduced with MSCV/ZFP_huKO and analyzed on EGFP expression on Day 3 and 17 after transduction. (C) EGFP expression was measured by RT-PCR in HEK293 cells analyzed with FACS (Fig. (A)). β-actin was used for a loading control.
Fig. 4. Epigenetic modifications of CMV and EF1-α promoter induced by ZFP809.
Notes: (A) DNAs obtained from EGFP-negative cells expressing pCMV_MLV/I/EGFP or pEF1_MLV/I/EGFP together with MSCV/ZFP_huKO were analyzed on DNA methylation of the promoters by bisulfite sequencing analyses (the left of the top or bottom panel). EGFP-positive cells expressing pCMV_dl587/I/EGFP or pEF1_dl587/I/EGFP were used as control (the right of the top or bottom panel). Open and closed circles represent unmethylated and methylated cytosines in the promoters, respectively (n = 9 or 10). (B) HEK293 cells were transfected with the pCMV/FLAG-ZFP809 together with transduction of MLV/EGFP or MSCV/EGFP. Genomic DNAs were isolated from the EGFP-negative fraction of the cells transduced with MLV/EGFP (the top panel) and the EGFP-positive fraction of the cells transduced with MSCV/EGFP (the bottom panel) were used for bisulfite sequencing analysis of the LTR. Open and closed circles represent unmethylated and methylated cytosines of the CpG dinucleotides in the LTRs, respectively (n = 9). (C) The cells expressing pCMV_MLV/I/EGFP or pCMV_dl587/I/EGFP were also analyzed on histone methylation around the promoter with ChIP-qPCR analyses using antibodies against H3k9me3 or IgG. The % input presents the enrichment of H3k9me3 relative to the control. Error bars show a standard deviation obtained from three independent experiments. Statistical P values were determined by the Student’s t-test. (D) Nuclear proteins obtained from the cells transduced with MLV/EGFP and MSCV/EGFP were precipitated with antibodies against H3K9me3 and mouse IgG as a control and the samples were used for PCR amplification using specific primers for the EGFP. The value of % input represents the enrichment fold of H3K9me9 compared to the control. Error bars show a standard deviation obtained from three independent experiments. Statistical P values were determined by the Student’s t-test.
