Strategy for targeting theHunkallele. A) Nucleotide sequence of mouse Hunk cDNA corresponding to targeted exon 4 (highlighted). Arrows mark exon/intron junctions. Subdomains VII (single-underline) and VIII (double-underline) of the catalytic domain are indicated. B, C Nucleotide sequences of Hunk cDNA with spliced out exon 4 (B) or exons 4 and 5 (C). Note, a frame shift and truncation protein truncation occurs following splicing between exons 3 and 5. However, splicing between exons 3 and 6 (which doesn’t induce a frame shift), produces a protein lacking subdomains important for kinase activity. D Schematic of the targeting strategy. Hatched boxes represent exons. EcoRI (R), XhoI (X), NotI (N) sites (for cloning “short” and “long arms” into pPNT vector) and BamHI (B) sites (for “long arm” recombination analysis) are shown. Arrows represent primers (used for ES clone analysis and genotyping of mice). Nucleotide positions are shown according to mouse chromosome 16 sequence GeneBank Acc. No. NT 039625 2. Neomycin phopshotransferase (dark grey arrow) and HSV thymidine kinase (black arrow) expression cassettes, are also shown. E PCR analysis of ES clones (primers MK1S and neoB) demonstrating homologous recombination of the “short arm” in clones 194, 292 and 328 but not in negative clone N or parental E14Tg2a ES cells (ES). F Southern blot analysis using P32-labeled probe “L” (white box in panel D) and BamHI-digested genomic DNA from clones 194 and 328, negative clone N and parental E14Tg2a ES cells (ES). G PCR genotyping of Hunk−/−(lane1), Hunk+/−(lane2) and Hunk+/+(lane3) mice (primers MK1A, MKK and neoB). H Scheme of exons 2 through 7 of Hunk cDNA, demonstrating amplification products from wild type allele (wt), targeted allele missing exon 4 (KODex4) and with additionally spliced out exon 5 (KODex4&5). Arrows represent RT-PCR Primers. I RT-PCR amplification from cerebellum RNA of Hunk+/+(lane1), Hunk+/−(lane2) and Hunk−/−(lane3) mice. MW – DNA molecular weight markers.