1. Volume 13, Issue 1, Pages 59-66 (January 2006)
Epigenetic Regulation of Lentiviral Transgene Vectors in a Large Animal Model 
Andreas Hofmann, Barbara Kessler, Sonja Ewerling, Andrea Kabermann, Gottfried Brem, Eckhard Wolf, Alexander Pfeifer 
Molecular Therapy 
Volume 13, Issue 1, Pages (January 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Transgene expression levels of different lentiviral integrants. (A–C) Identification of individual proviruses from three founder (F0) animals—8507 (male, two integrants), 8514 (female, three integrants), and 8506 (male, one integrant)—and their offspring (8774–8783, 8784–8793, and 9031–9036, respectively) by Southern blot analysis using BamHI, which cuts only once in the proviral genome. Six individual proviruses (a–f) were segregated by mating of the three transgenic founders. (D–F) Western blot analysis of transgene expression in extracts of skin biopsies (top). β-Actin antibodies were used for loading control (bottom). (G) Mean eGFP fluorescence of animals carrying integrants a–f (black columns) compared to their founders (white columns). Expression levels were calculated relative to the actin loading control. (H) Southern blot analysis using SstI, which cuts in the LTRs of the provirus (top). All animals exhibit the 3.4-kb full-length proviral insertion, demonstrating the intactness of the integrated provirus (bottom). Triangle, self inactivating mutation; PGK, phosphoglycerate kinase promoter; eGFP, enhanced green fluorescent protein; LTR, long terminal repeat; cPPT, polypurine tract; WPRE, woodchuck hepatitis responsive element. Animals carrying single integrants are underlined.
Molecular Therapy  , 59-66DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Correlation between methylation status of lentiviral integrants and transgene expression levels. (A–C) Analysis of the methylation status of the promoter region using the methylation-sensitive restriction enzyme AscI. (A, B) Unmethylated DNA yields a 1.5-kb band after EcoRI/AscI digestion. Methylation of the CpG dinucleotide present in the AscI site results in a 1.8-kb band in the Southern blot. (C) The methylation status of integrants a–f was quantified by calculating the ratio of methylated to unmethylated bands. (D–F) Methylation of the coding region was analyzed by digesting genomic DNA with the methylation-sensitive HpaII (H) and its methylation-insensitive isoschizomer MspI (M). (F) Methylation status of integrants a–f as quantified by calculating the ratio of MspI to HpaII bands: (M − H)/H (M, MspI band intensity; H, HpaII band intensity). (G) Analysis of eGFP expression by Western blotting of skin biopsies. Mean transgene expression levels in F1 animals, as calculated by comparing individual bands with the band intensity of a recombinant eGFP standard, relative to the actin loading control. (H) Correlation between mean eGFP expression and methylation levels in the promoter region (filled circles) and the coding region (open circles) of individual integrants (a–f).
Molecular Therapy  , 59-66DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
High-resolution DNA methylation analysis of CpG dinucleotides of the proviral PGK promoter and the eGFP gene by bisulfite genomic sequencing. (A) Methylation pattern of a 234-bp region (24 CpG islands; top) in the PGK promoter of animal 8778 carrying integrant b (upper middle), animal 8779 carrying integrant a (lower middle), and animal 8791 carrying integrant e (bottom). The AscI recognition site, which was used for Southern blot analysis, is indicated. (B) Mapping of the methylation status of the eGFP cassette (255 bases, 18 CpG sites) of animals 8778 (upper middle), 8779 (lower middle), and 8791 (bottom). Closed circles indicate methylated CpGs, open circles indicate unmethylated CpGs.
Molecular Therapy  , 59-66DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Analysis of transgene expression in skin fibroblasts isolated from lentiviral transgenic pig (A) Cells were treated for 2 days with DMSO, 5-azaC (Aza), or TSA (TSA). Thereafter, the cells were incubated for 3 days in medium (∅), in the presence of DMSO (DMSO), or in the presence of TSA (Aza/TSA). After 5 days, cells were analyzed by FACScan and methylation-sensitive Southern blot analysis. (B–E) Representative FACScan analyses of transgenic fibroblasts. Cells were treated with (B) DMSO, (C) 5-azaC, (D) TSA, or (E) 5-azaC and TSA. Overall mean and geometric mean fluorescence values are given. Numbers under the gate refer to % eGFP+ cells. (F) Statistical analysis of the effect of 5-azaC, TSA, or consecutive 5-azaC/TSA treatment on mean fluorescence. DMSO-treated cells (as control) were set as 100%. Geometric means ± SEM (n = 7 experiments, *P < 0.05 versus control) are given. (G) Analysis of proviral methylation status using the methylation-sensitive restriction enzyme AscI. Unmethylated DNA yields a 1.5-kb band, methylation of the restriction site will result in a 1.8-kb signal. (H) The methylation level of skin fibroblasts was quantified by calculating the ratio of methylated to unmethylated bands.
Molecular Therapy  , 59-66DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions