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. 2015 Sep 3;5(4):558–568. doi: 10.1016/j.stemcr.2015.08.005

Figure 2.

Figure 2

Correction of Tcirg1 Mutation in oc/oc iPSCs

(A) Upper lane: schematic representation of the BAC used for the gene targeting, containing the 11-kb Tcirg1 locus plus its 5′ and 3′ flanking regions (55 and 78 kb, respectively), as well as CmR and HIS3 genes belonging to the BAC backbone. Middle and lower lanes: schematic representation of the 1.6-kb deletion in genomic DNA present in oc/oc mutants compared with the WT genomic region. oc/oc mice are homozygous for the mutation.

(B) Experimental outline for generating gene-corrected oc/oc iPSC clones (see text for details). Only clones containing and expressing corrected Tcirg1, as assessed by PCR on genomic DNA and by RT-PCR, were subjected to FISH and karyotype analyses.

(C) Representative metaphase spreads after FISH using the BAC as probe (red), which hybridizes with the BAC-inserted fragment as well as with the mutated form of Tcirg1. White arrows indicate the presence of the probe only on the two 19 chromosomes in clones 18-BAC, 28-BAC, and 32-BAC, in a sub-centromeric localization corresponding to the endogenous Tcirg1 location. Since these clones were previously screened by PCR for the presence of the correct Tcirg1 gene, the presence of two signals in the expected position suggests that at least one of the two alleles was targeted. Chromosomes were counterstained by DAPI (blue). Scale bars, 5 μm.

(D) RT-PCR analysis for detection of Tcirg1 expression in the WT 39 iPSC clone (used as a positive control), in the oc/oc 74 iPSC clone (negative control), and in the three corrected oc/oc iPSC clones, using the primers represented by red arrows in (A). Mouse Gapdh was used as housekeeping control.

(E) PCR analysis for detection of HIS3 and CmR backbone sequences into the genome of iPSC clones. First lane: the positive control showing the PCR product obtained from the BAC; second lane: the WT iPSC 39 clone used as a negative control; third and fourth lanes: 18-BAC and 32-BAC devoid of the tested backbone sequences. These sequences were absent also in 28-BAC, which was tested separately (data not shown). The last two lanes show a 168-bp fragment of the CmR and a 191-bp fragment of HIS3 gene obtained in two clones previously discarded due to the BAC insertion in other chromosomes.

See also Figure S2.