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Fig. 4. Mitfmi-rw/mi-rw mutant mice carry a genomic deletion
in Mitf encompassing the exons 1H, 1D and 1B. (A)
Phenotype of an Mitfmi-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 Mitfmi-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.
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