Fig 1.
Targeted genome editing in RS-SCID fibroblasts.
(A) Schematic of genome editing strategy. Homology-directed repair (HDR) between the prkdc locus and the donor DNA is promoted by ZFN cleavage in intron 84 (BS, binding site). The HDR donor consists of flanking homology arms (dashed lines), splice acceptor (SA), cDNA encoding prkdc exons 85 and 86, polyadenylation signal (pA), neomycin resistance cassette (NeoR). The SCID underlying mutation in exon 85 (mut*), and primer binding sites for PCR analysis (5’-junction J5-F/J5-R; 3’-junction J3-F/J3-R; allelic discrimination AD-F/AD-R; mRNA expression RT-F/RT-R) are indicated. (B) Genome editing. After transfection of SCID fibroblasts with various ratios of donor DNA to ZFN expression plasmids, successful gene targeting in polyclonal samples was detected by an inside-out PCR amplification of the genome–donor 5´-junction (J5-F/J5-R). (C) Expression of corrected prkdc mRNA. After transfection of SCID fibroblasts, successful splicing from exon 83 to cDNA was detected with an inside-out RT-PCR strategy using primers RT-F/RT-R.
Fig 2.
Functional correction of RS-SCID fibroblasts.
(A) DNA-PK dependent phosphorylation of RPA2. Treatment of fibroblasts with camptothecin (CPT) induces DNA-PK dependent phosphorylation of RPA2, which was detected by Western blot analysis using an RPA2 specific antibody. Detection of ß-actin served as a loading control. Positions of RPA2 and its phosphorylated form, RPA2-P, are indicated on the right. (B) Rescue of radiosensitivity. Fibroblasts were cultured with increasing amounts of the radiomimetic drug bleomycin. Cellular sensitivity to the drug was quantified by counting number of surviving colonies relative to untreated samples. Data are represented as mean ± SD (N = 3). 3T3, NIH-3T3 fibroblasts; Fib.S, SCID fibroblasts; Fib.T, gene targeted SCID fibroblasts; Fib.D, fibroblasts treated with randomly integrated donor.
Fig 3.
Evaluation of pluripotency of generated iPSCs.
(A) Pluripotent stem cell marker gene expression. Oct3/4, Nanog, Zfp42, Esg1, Eras, and fgf4 mRNA expression was determined by qualitative RT-PCR (see S2 Table). Housekeeping gene 36B4 (+/- reverse transcriptase) were included as controls. ES.CCE, murine embryonic stem cell line; Fib.S, SCID fibroblasts; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, gene targeted SCID iPSC clones; iPS.WT, wild-type iPSC clone. (B) In vivo differentiation analysis. Teratoma formation was induced by subcutaneous injection of iPSCs into mice. Hematoxylin/eosin-stained sections of teratoma-derived from clones iPS.S6 and iPS.T25 are shown. (C) Karyotype analysis. Spectral karyotyping (SKY) was performed to detect microscopic genomic abnormalities, translocations and aneuploidies in untreated or genetically corrected SCID iPSC clones. SKY analysis of clone iPSC.T25 is shown (see also S2 Fig).
Fig 4.
Targeted genome editing in SCID-derived iPSCs.
(A) Verification of gene targeting. Inside-out PCR strategies (see Fig 1A) were used to verify correct 5´ (J5) and 3´-junctions (J3) of the integrated donor. Allelic discrimination (AD) PCR was used to assess mono- vs. bi-allelic integration. Targeted allele runs at 2.99 kb. Sizes of all expected PCR amplicons are indicated on the right. iPS.WT, wild-type iPSC; iPS.S6, SCID iPSC clone; iPS.T8, iPS.T25, iPS.T44, iPS.T45 and iPS.T60, targeted SCID iPSC clones. (B) Expression of corrected prkdc mRNA. Successful splicing from exon 83 to cDNA encompassing exons 84/85 was detected by an inside-out RT-PCR strategy using primers RT-F/RT-R.
Fig 5.
In vitro differentiation of iPSCs to proT-cells and T-cells.
(A) Schematic of in vitro T-cell differentiation from iPSCs. Differentiation of iPSCs starts with formation of embryoid bodies that are dissociated to give rise to hematopoietic stem and progenitor cells (HPC). DL-1 mediated Notch signaling coaxes HPC development towards early proT-cells (DN2), which undergo DNA-PK dependent V(D)J recombination. After passing through DN3 and DN4 stages, preT-cells mature into double-positive (DP) T-cells that express the beta chain of the T-cell receptor (TCRß). Dashed lines indicate to what stage iPSC clones are expected to differentiate. (B) Assessment of T-cell differentiation. In vitro T-cell differentiation was analyzed by flow cytometry after two weeks of co-cultivation on OP9-DL1. Gating (indicated on top of each column) was applied in the following order: FSC/SSC and CD45+/DAPI—to assess CD4/CD8 expression; CD8–/CD4—to gate for DN1-DN4 stage cells; CD8–/CD4– (DN) or CD8+/CD4+ (DP) to assess TCRß expression. Numbers indicate percentage of cells in each quadrant. HPC, lineage-negative bone marrow cells; iPS.WTX, wild-type iPSC; iPS.S6X, SCID iPSC clone; iPS.T25, gene targeted SCID iPSC clone.
Fig 6.
Polyclonal T-cell receptor recombination.
In vitro generated T-cells were analyzed by spectratyping, i.e. quantitative RT-PCR expression analysis of the variable beta chains. Exemplarily shown are results for Vß1, Vß8.3 and Vß10 (see also S5 Fig). X-axis depicts detected PCR fragment size in bp, Y-axis depicts counts of obtained PCR fragments. Thymus, T-cells isolated from thymus as a positive control; HSC, in vitro generated T-cells from lineage-negative bone marrow cells; iPS.WTX, wild-type iPSCs; iPS.S6X, SCID iPSC clone; iPS.T25X, gene targeted SCID iPSC clone.