Inactivation of the tyrosinase locus in C57BL/6NTac ES cells and mice.

<p>(A) Gene targeting scheme for the tyrosinase locus. A point mutation was introduced in exon 1 (C103S) of the tyrosinase allele. The Puromycin selection marker (PuroR) is shown as a grey box. Numbered dashes indicate external probes used for Southern Blot analysis of ES cell clones, lettered arrows indicate oligos used for genotyping of mice. Deletion of the F3 flanked selection marker was achieved in vitro in targeted ES cell clone A-B6. (<b>B</b>) Southern Blot validation with genomic DNA isolated from 6 ES cell clones and wildtype C57BL/6 genomic DNA as a control (W) using external probe 1 in combination with EcoRI restriction digest (upper panel, Probe 1, E) leading to a wildtype allele of 4,8 kb (Wt) and a targeted allele of 7,7 kb (tm) and confirmatory Southern Blot validation with external probe 2 in combination with BglI restriction digest (lower panel, Probe 2, B), leading to a wildtype allele of 12,7 kb (Wt) and a targeted allele of 17,7 kb (tm). (<b>C</b>) PCR verification. Clone A-B6 was selected for chimera generation and germline transmission. Primer combinations f + g (upper panel) applied on genomic DNA displayed a 151 bp wildtype <i>Tyr</i> allele in BALB/c control mice (lane 1, Balb/c), C57BL/6 control mice (lane 2, C57BL/6) and in chimeras generated with C57BL/6 ES cells injected in homozyogus A++ host embryos (lane 4, A++/B6 Ch). In addition the modified <i>Tyr^tm1</i>Arte allele was detected in homozygous Albino A++ mice (lane 3, A++) and in chimeras generated with C57BL/6 ES cells injected in homozyogus A++ host embryos (lane 4, A++/B6 Ch). Lower panel: the introduced point mutation was verified by sequencing both strands of the PCR fragment amplified with primers d + e.