Lymphoid Regeneration from Gene-Corrected SCID-X1 Subject-Derived iPSCs
TALEN-Mediated Gene Correction and Lymphoid Differentiation of SCID-X1 iPSCs
(A) Schematic representation of the IL-2Rγ gene, the endogenous target locus of our subject-specific mutation, and the corrective donor vector used. The sequence of the subject-specific IL-2Rγ splice-site target mutation and the corrective donor vector sequence are also shown below. The point mutation causing the alteration of this exon/intron consensus splice site is indicated in red. Exon 3 sequence immediately preceding the splice site is denoted by green, with bases in bold. TALEN binding sites are indicated by black arrows.
(B) Identification and isolation of corrected iPSCs through single-cell clonal amplification and screening of the PCR products with an XmaI restriction digest that is specific to the correction event. Corrected clones and subclones are identified by (#).
(C) Chromatogram of the corrected iPSC clone indicated in Figure 1 B, as verified by sequencing. The red boxes indicate each of the silent mutations that were introduced to abolish TALEN activity on the corrective or corrected DNA sequences. Black box indicates the corrected disease-causing base.
(D) Comparative analysis of FACS data from wild-type, SCID-X1 mutant, and SCID-X1 corrected iPSCs. Data show CD34 and CD43 expression at day 13 of differentiation. Isotype controls are included in the left panel.
(E) RT-PCR analysis of RNA extracted from T cell precursors in the floating fraction generated from wild-type, subject-derived SCID-X1 mutant iPSCs, or SCID-X1 corrected iPSC clones.
(F) FACS analysis of the floating fraction of cells co-cultured on OP9-DL feeders from two independent wild-type, SCID-X1 mutant, and SCID-X1 gene-corrected iPSC lines. CD45+ and CD45+/CD4+/CD8a+ populations are indicated.