Targeted knock-in of IDH1R132H/WT hotspot mutation in a human cancer cell line. (A) To faithfully recapitulate expression of heterozygous IDH1R132H/WT mutations as observed in human tumors, a targeting vector was designed to introduce the IDH1R132H mutation in one endogenous allele of IDH1 in HCT116. Relative genomic positions of exons are indicated, including 5′ UTR (white boxes) and coding sequences (black boxes). Homology arms (HAs) were cloned from HCT116 parental cells and are shown in red. The left HA (LHA) was altered by site-directed mutagenesis to create the IDH1R132H mutation (indicated by yellow star). The homology arms flank a synthetic exon promoter trap (SEPT) cassette. The promoterless SEPT element contains a splice acceptor (SA), internal ribosomal entry sequence (IRES), neomycin selectable marker (neo), and polyadenylation site (pA), which are flanked by loxP sites (green triangles). Inverted terminal repeats (ITR) of the adeno-associated virus (AAV) vector flank the homology arms. Correctly targeted alleles result in incorporation of the SEPT cassette along with the R132H mutation. Targeted clones were infected with Cre adenovirus to excise the selectable element, generating a clone that differs from the parental cell line by the single base pair mutation in exon 4 and 34-bp loxP scar in the adjacent intron. (B) Sequencing validation of IDH1R132H/WT knock-in clones. Representative sequencing chromatograms for IDH1 codons 131–133 in genomic DNA (left) and cDNA (right) of HCT116 parental cells (top) and IDH1 knock-in cells (bottom). Knock-in clones contain a heterozygous G>AG mutation at chr2:208,938,618 and are heterozygous for wild-type allele (CGT) and mutant allele (CAT) coding for an arginine (R) to histidine (H) change at amino acid 132. Measurement of D-2-hydroxyglutarate (D-2-HG) in (C) clarified cell lysate and (D) cell culture medium over cells, collected after 48 h incubation. Shown is the mean ±SD of triplicate measurements.
