Generation of a conditional humanized mutant CALR knockin mouse model. (A) Diagrams showing the endogenous mouse (Ms) CALR gene locus (CALR⁺), the targeting construct, the targeted conditional knockin allele (CALR^fl), and the recombined allele (CALR^del) after Cre recombination to remove the PGKNeopAstop cassette. The recombined allele is designed to carry the mouse CALR genomic region, including mouse exons 1 to 7 followed by the 5′ part of mouse exon 8 fused to the 3′ part of the human (Hu) mutant (mut) CALR cDNA sequence (orange). LoxP sites are indicated by red arrows. (B) Amino acid alignment of WT and mutant CALR proteins. There is a 94% identity between the entire amino acid sequences of human and mouse proteins. (Top panel) Alignments show amino acid 343 to the C terminus of human mutant CALR and the designed humanized mutant CALR knockin product. (Bottom panel) The predicted product if the corresponding 52-bp region is deleted in the mouse CALR gene. Alignment was performed by using an online program available at www.expasy.org. Sites of the mouse/human junction and the 52-bp deletion are indicated. (C) Diagram showing location of the primers for PCR genotyping. F and R indicate forward and reverse primers. (D) Characterization of ES cell targeting. PCR performed on genomic DNA from ES clones (lanes 1-5) using primers F and R shown in (C). The ES clone in lane 5 is correctly targeted. (E) Characterization of Cre/LoxP-mediated recombination of targeted ES cells. PCR was performed on genomic DNA from ES cells upon Cre recombinase expression by using primers F and R as shown in panel C. fl/+, targeted ES clone; +/+, WT ES clones; lanes 1 and 2, Cre recombined ES clones. (F) Sequencing traces of RT-PCR product showing correct humanized mutant CALR expression with the 52-bp deletion indicated (52bp del). RT-PCR was performed on total RNA from an ES clone that was Cre recombined, and the PCR product was cloned and sequenced. M, DNA marker; UTR, untranslated region.