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SUPPLEMENTAL FIGURE 1
Disruption of the mouse Epha2 locus on chromosome 4 by homologous recombination 17, 37. (A)
Schematic showing the disruption site in exon 5, and location of PCR genotyping primers (Table
S1). (B) PCR amplification across the 5´ and 3´ boundaries of the disruption site in genomic
(g)DNA (lanes 1-4), and in cDNA following reverse transcription (lanes 5-8), respectively. Lane
2 confirms the 5´-disruption boundary in gDNA, and lane 8 confirms the 3´-disruption boundary
in cDNA. (C-D) Sequence analysis of PCR amplicons from panel B that cross the 5´ and 3´ ends
of the disruption site. (C) Wild-type 5´-end showing reading frame flanking an endogenous
HindIII site in exon-5 (B, lane 1). (D) Disrupted 5´-end showing change in reading-frame after
the HindIII site (B, lane 2) at codon 428 from the translation start codon (ATG). This is predicted
to result in a nonsense mutation (p.F428LfsX85) with the addition of 84 novel amino acids
before premature termination of protein translation at an in-frame translation stop-codon (TGA)
located 85 codons downstream within the targeting vector (data not shown). (E) Wild-type 3´end showing reading frame for correct splicing of transcript between exon-5 and exon-6 (B, lane
5). (F) Disrupted 3´-end showing incorrect splicing of transcript between the targeting vector
(neo cassette) and exon-6 (B, lane 8). Mouse tail genotyping, lens total RNA extraction, reverse
transcript (RT)-PCR, and DNA sequencing were performed as described 38.