Fig. 4. Mitf^mi-rw/mi-rw mutant mice carry a genomic deletion in Mitf encompassing the exons 1H, 1D and 1B. (A) Phenotype of an Mitf^mi-rw/mi-rw mutant mouse. Generally, coat pigment patches and eye size are not correlated, as eye size is determined by the development of the RPE and coat patches by neural crest-derived melanocytes. To the right is shown the DNA sequence flanking the genomic deletion, with the numbers of the first and last base referring to the positions on chromosome 6 according to assembly NCBIM36. The schematic beneath shows the extent of the deletion and highlights the novel splice junctions that are generated between the upstream exons 1A, 1J, 1C, 1MC and 1E and the downstream exon 2A. For details, see text. (B) RT-PCR using RNA from E12.5 wild-type and Mitf^mi-rw (rw) mutant embryos and the corresponding P0 newborns. Note increased band intensity in rw with exons 1A, 1J and 1E, but decreased band intensity at E12.5 with pan-specific primers (exon 9). (C) Real-time PCR from RNA of whole eyes harvested at the indicated ages, using primers as in B. The results are expressed as RNA levels in rw mutants relative to those in corresponding wild-type embryos (groups of 14 eyes each, three measurements each in triplicate). The increases in rw over wild type in 1A and 1J, as well as the decrease in exon 9 at E12.5, are statistically significant (P<0.01, Student's t-test). P-values for the increase in 1E are <0.02 at E12.5 and =0.13 at P0. No significant difference was found for exon 9 at P0 (P=0.27). (D) RT-PCR using primers spanning four exons. Note the differently sized products in wild type and rw for isoform 1A. For isoform 1J, the expected larger product in wild type is not seen because its relative level is low (see Fig. 2), but the smaller sized product in rw is visible. In 1E-4, the white arrowhead points to the correct E-Mitf band.