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STEM_718_sm_SuppFig1.pdf227KFigure S1. Characterization of parental and integration-free parental iPS lines A. Phase contrast image of iPS clone II grown on feeder cells and immunofluorescence images of feeder-free cultures. B. RT-PCR assays to assess expression of pluripotency-associated genes in looped-out iPS clones C. H&E staining of teratoma derived from iPS clone II showing tissues representative of all three germ layers. D. Immunofluorescence images of looped-out clones stained for pluripotency-associated transcription factors and surface markers in feeder-free cultures.
STEM_718_sm_SuppFig2.pdf142KFigure S2. Targeting of clone IV and analysis of donor template integration events A. Southern blot using a 5′ external probe on genomic DNA digested with PvuII. A betaglobin allele that has not undergone gene targeting gives a 12.8kb band while a targeted allele gives an 8kb band due to the presence of a PvuII site in the neomycin-resistance gene. Asterisks indicate gene-targeted clones while clones not marked with an asterisk are negative controls. B. Southern blots for assessing off-target integrations of the donor construct using a probe hybridizing to the 5′ homology arm of the donor construct on genomic DNA digested with either BamHI or XmnI. The expected size for correct gene targeting events is 1.7kb for BamHI digestion and 5.5kb for XmnI digestion. Clones with asterisks were determined to be correctly targeted (as determined by PCR and Southern blot – see Figure 3) while clones without asterisks were those that became negative after further expansion in culture.
STEN_718_sm_SuppFig3.tif837KFigure S3. Analysis of off-target effects A. DNA sequencing of closely-related delta and gamma-globin paralogue genes in successfully targeted iPS cell clones. Segments of DNA sequence from the beta-globin locus are shown with zinc finger binding sites highlighted in yellow for both ZFN sites 1 and 2. Corresponding sequences from the delta-, a-gamma- and g-gamma-globin loci are also shown with bases differing from the beta-globin locus highlighted in red. DNA sequences of these paralogous loci in targeted clones are shown below. B. DNA sequencing of closely related, computationally identified potential off-target ZFN cleavage sites. The intended beta-globin target sites of ZFN pairs A, B, and C used for gene correction are shown with binding sites highlighted in yellow. Sites in the human genome that differ from the ZFN target site by only one bp are shown with the altered base in red with the genomic location for each site provided to the left. Unaltered sequences of these sites in successfully corrected clones IA13, IA38 and IB49 are shown below each site.
STEM_718_sm_SuppFig4.pdf179KFigure S4. Characterization of integration-free gene corrected clones A. Immunofluorescence images of iPS clones stained for pluripotency-associated transcription factors and surface markers in feeder-free cultures. B. RT-PCR analysis to assess expression of pluripotency-associated genes. C. Immunofluorescence images of iPS lines differentiated in vitro. D. Telomerase activity assays.
STEM_718_sm_SuppTable1.tif104KTable S1. Characterization of parental iPS clones. Karyotype was determined by SKY-FISH; underlined: correct diploid chromosome spreads (chromosome number, sexchromosomes, number of spreads shown in brackets); non-underlined: non clonal aberrations (number of observed chromosomes, extra or missing chromosomes are shown in bold and the number of spreads with the specified chromosome set are in brackets). In vitro differentiation: cells were differentiated via embryoid body (EB) formation for 8 days followed by culture of EB on gelatin-coated plates for 2 more weeks in media containing 20% FBS.
STEM_718_sm_SuppTable2.tif175KTable S2. Characterization of gene corrected iPS clones. Karyotype was determined by SKY-FISH; underlined: correct diploid chromosome spreads (chromosome number, sex-chromosomes, number of spreads shown in brackets); non-underlined: non-clonal aberrations (number of observed chromosomes, extra or missing chromosomes shown in bold, and the number of spreads with the specified chromosome set shown in brackets). In vitro differentiation: cells were differentiated via embryoid body (EB) formation for 8 days followed by culture of EB on gelatin-coated plates for 2 more weeks in media containing 20% FBS.
STEM_718_sm_SuppTable3.tif136KTable S3. Characterization of integration-free parental and gene corrected iPS cell lines. Cell line names, ZFN pairs used for targeting, and molecular characterization performed are shown for all gene targeted iPS cell clones described in this study. n.d. indicates that it was not possible to determine the allele targeted due to a mixed population of cells. “n.a. ” indicates that the assay or procedure was not performed.
STEM_718_sm_SuppInfo.pdf152KSupporting Information

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