Figure 1. Targeted Correction of the Expanded Allele in HD-iPSCs.

(A) Exon 1 (e1) and surrounding regions of the expanded HD locus of patient-derived HD-iPSCs containing 72 polyglutamine repeats; the designed targeting vector from BAC RP11-866L6 with short and long arms of 4.5 and 10 kb respectively; and the resulting correctly targeted allele with 21 polyglutamine repeats and FLP flanked PGK-neo and EGFP cassettes inserted upstream of the reported promoter region. HindIII sites (H) and resulting targeted fragment sizes using the 300 bp external Southern probe (black bar) are shown.

(B) Brightfield (BF) and GFP fluorescent images of HD-iPSCs nucleofected with pEGFP plasmid at 1 day postnucleofection and after 3 weeks of G418 selection.

(C) Primary PCR-based screen of G418 resistant clones for loss of the expanded (72 CAG) allele. Targeted clones indicated in green were later verified by Southern blot as correctly targeted alleles.

(D) Southern blotting analysis of HindIII digested HD-iPSC clone genomic DNA and control samples. A P32 radiolabeled 300 bp external probe hybridizes with a 10 kb nontargeted (WT) or 5 kb targeted HindIII. Labeled are positive and negative BAC DNA controls for cassette insertion or noninsertion (BAC Rec+ and BAC Rec−, respectively); nontargeted HD-iPSC clones C99 and C112; and correctly targeted HD-iPSC clones C127 and C116. For further Southern blot analysis and sequencing of clones, see Figure S1.

(E) Expanded polyglutamine correction revealed by western blot analysis of lysates from HD-iPSCs and corrected clones C116 and C127 using antibodies for huntingtin (Htt2166) and expanded polyglutamine (1C2). A line of normal iPSCs (Park et al., 2008) derived from non-HD patient fibrobasts is included as a wild-type allele control.

(F) Summary of the gene targeting experiment and resulting targeting frequencies observed.