The cHS4 insulator block activation of Map17 but not Tal1.

<p>A: PCR analysis demonstrating insertion of 5 cassettes at RL5 in each orientation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#s4" target="_blank">methods</a>). At least 2 clones in each orientation are shown. B and C: Schematic of the structure of RL5 loci in the presence of the various tested cassettes in each orientation, and histograms illustrating the average activation (±standard deviation) of the flanking genes (relative to the control cDNA cassette). D: FACS Analysis: Whisker plots of the mean linear fluorescence of 5 to 15 clones containing cassette 234-β-EGFP flanked on either sides or on both sides by the 2.4 kb cHS4 insulator. Presence of one insulator decreases EGFP expression by more than 2-fold. Presence of 2 insulators has an even more pronounced effect. E: Histograms illustrating a Q-PCR analysis of EGFP expression of the clones analyzed by FACS in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#pone-0005956-g003" target="_blank">Figure 3A</a>. The RT-PCR results are similar to the FACS results. EGFP expression was normalized to expression of the β-2-microglobulin gene. The effect of the insulator was independent of its location within the cassette and of the orientation of the cassette in the locus.</p

primer used for RT-PCR.

<p>primer used for RT-PCR.</p

Orientation-dependent silencing of β-EGFP expression in the presence of the G8 repeats.

<p>A: Dot-plots illustrating EGFP expression of representative clones one or three months after RMCE. X-axis: forward-scatter; y-axis: FL-1 (EGFP) fluorescence. The horizontal line represents the level of auto-fluorescence of non-tranfected control MEL cells. Presence of one copy of G8 3′ of EGFP, or of two flanking copies of G8 caused silencing of the transgene in the N but not in the P orientation. B: Q-RT-PCR analysis of EGFP expression demonstrating that the silencing induced by the G8 repeats occurs at the mRNA level.</p

Insertion of hHS4, hHS5 and hG8 repeat do not block activation of Tal1 and Map17.

<p>A: PCR analysis demonstrating insertion of the various cassettes. B and C: Diagram illustrating the structure of the RL5 region after insertion of the various cassettes and histograms summarizing Q-RT-PCR determinations of the average fold increases (±standard deviation) of the flanking genes relative to the cDNA control cassette. The three cassettes tested had minimal effects on expression of Tal1 and Map17. The black bars represent the fold increase of the 234-β-EGFP cassette with and without cHS4 which was used as a control in this experiment. D and E: FACS and Q-RT-PCR analyses of EGFP expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#pone-0005956-g003" target="_blank">Figure 3D and 3E</a>) when cassettes 234-β-EGFP plus hHS4, hHS5 or G8 were inserted at RL5. Levels of expression in the presence of HS5, HS4 and G8 are respectively lower or higher than the controls both at the protein and mRNA levels.</p

Insertion of cassette 234-β-EGFP activates genes near the RL5 integration site:

<p>A: Structure of the region around the RL5 integration site on chromosome 4. The integration site RL5 is located at Chr 4: position 114756771 (mouse build July 2007 (mm9) assembly). B: Schematic of the RMCE reactions. The numbers above the gene represent the average increase in levels of expression of the flanking genes after insertion of the 234-β-EGFP cassette at RL5. Both orientations are represented. The black triangle represents the two inverted Lox sites. Fold increases were calculated relative to the β-2-microglobulin gene and relative to the expression of the same gene when the control cDNA cassette was inserted at the same locus (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005956#s4" target="_blank">methods</a>). C: Histogram summarizing the increase of the flanking genes (±standard deviation).</p

DNA segment inserted in the various cassettes used in this study.

<p>The cHS4 insulator used in this study contained 2 tandem duplicated copies of U78775.</p

Complete Genome Phasing of Family Quartet by Combination of Genetic, Physical and Population-Based Phasing Analysis

<div><p>Phased genome maps are important to understand genetic and epigenetic regulation and disease mechanisms, particularly parental imprinting defects. Phasing is also critical to assess the functional consequences of genetic variants, and to allow precise definition of haplotype blocks which is useful to understand gene-flow and genotype-phenotype association at the population level. Transmission phasing by analysis of a family quartet allows the phasing of 95% of all variants as the uniformly heterozygous positions cannot be phased. Here, we report a phasing method based on a combination of transmission analysis, physical phasing by pair-end sequencing of libraries of staggered sizes and population-based analysis. Sequencing of a healthy Caucasians quartet at 120x coverage and combination of physical and transmission phasing yielded the phased genotypes of about 99.8% of the SNPs, indels and structural variants present in the quartet, a phasing rate significantly higher than what can be achieved using any single phasing method. A false positive SNP error rate below 10*E-7 per genome and per base was obtained using a combination of filters. We provide a complete list of SNPs, indels and structural variants, an analysis of haplotype block sizes, and an analysis of the false positive and negative variant calling error rates. Improved genome phasing and family sequencing will increase the power of genome-wide sequencing as a clinical diagnosis tool and has myriad basic science applications.</p></div

Characterization of γ-globin expression variability in clonal sibling baso-E populations.

<p>(A) Experimental design. BM CMPs (box) from control individuals were directly sorted into AFT024 coated 96-well plates at a concentration of one cell per well. After 4 days of culture, progeny from a single CMP were divided into two sister wells and allowed to differentiate in parallel for 10 additional days. RNA from these clonal sibling baso-E populations was then collected for globin analysis. (B) Levels of γ-globin expression are not determined at the CMP level. Percent γ-globin expression was determined by qRT-PCR analysis. Error bars represent the standard deviation between at least three independent cDNA and qRT-PCR replicates. Student’s t-test was used to determine statistically significant differences in γ-globin expression between sister cultures. Levels of γ-globin expression was significantly different in 5 out of 14 sister cultures tested.</p

Variability of γ-globin expression in response to HU in clonal cultures of baso-E generated from BM CMPs.

<p>(A) Clonal populations of baso-E generated from unicellular BM CMPs from two, unrelated female non-sickle donors (donor 1; donor 2) were generated by co-cultured on AFT024 with or without 15uM HU for 14 days. RNA was extracted and qRT-PCR were performed. Expression of γ-globin expression was calculated as 100*γ/(γ+β). Error bars represent the standard deviation of at least three replicates of the reverse-transcription and qRT-PCR reactions, clonal averages shown. Intra-donor variation between clonal populations is shown at baseline (top panel) and after treatment with HU (bottom panel). (B) Intra-donor γ-globin expression from pooled baseline or HU dosed clones. Error bars represent standard error between clones in dose status group. Student’s t-test was used to determine significant differences in intra-donor γ-globin expression in response to HU. Values p<0.05 are denoted. (C) Intra-donor pooled clones by dose status were evaluated for expression of markers for erythroid differentiation. Student’s t-test with p<0.05 was used to determine significance.</p

Inter and intra-individual variation in γ-globin expression in baso-Es produced in the presence or absence of AFT024.

<p>(A) Variation in HbF levels are recapitulated in culture. Peripheral blood mononuclear cells (PBMCs) from four control donors (left panel) and four sickle cell donors (right panel) were differentiated <i>in vitro</i> into populations of baso-Es and globin expression was determined by HPLC. Baso-Es were collected on day 14 of culture system. Percent γ-globin was calculated as ((100*(Gγ + Aγ)/(Gγ + Aγ + β)). Error bars represent standard deviation in γ-globin levels over independent biological replicates. (B) Co-culture of CD34+ cells with AFT024 stromal cells decrease background γ-globin expression. CD34⁺ PBMCs from two control donors were isolated, split into two fractions and differentiated into baso-Es <i>in vitro</i> in the presence or absence of mouse embryonic liver stromal cells (AFT024). On day 14, baso-Es were collected for HPLC analysis. Representative chromatograms of globin expression obtained by HPLC analysis from circulating red blood cells <i>in vivo</i> (left chromatograms), day 14 baso-E populations derived <i>in vitro</i> from CD34⁺ PBMCs without AFT024 stromal cell co-culture (middle chromatogram) or with AFT024 co-culture (right chromatogram). (C) Histogram illustrating the HPLC quantification of the effect of AFT024 on γ-globin expression. Average percent γ-globin shown, error bars represent percent γ-globin variation between donors. (D) Clonal population of baso-E exhibit large variation in γ-globin expression. Individual Common Myeloid Progenitors (CMPs) were isolated from a control bone marrow (BM) donor and placed in culture with or without AFT024 cells to generate clonal populations of baso-Es. After 14 days of culture, the percent of γ-globin expression (γ/γ+β) was determined by qRT-PCR analysis. Error bars represent the standard deviation in average γ-globin expression between replicate rounds of qRT-PCR.</p