An Unstable Targeted Allele of the Mouse <i>Mitf</i> Gene With a High Somatic and Germline Reversion Rate

Abstract

The mouse Mitf gene encodes a transcription factor that is regulated by serine phosphorylation and is critical for the development of melanin-containing pigment cells. To test the role of phosphorylation at a particular serine, S73 in exon 2 of Mitf, we used a standard targeting strategy in mouse embryonic stem cells to change the corresponding codon into one encoding an alanine. By chance, we generated an allele in which 85,222 bp of wild-type Mitf sequence are duplicated and inserted into an otherwise correctly targeted Mitf gene. Depending on the presence or absence of a neomycin resistance cassette, this genomic rearrangement leads to animals with a white coat with or without pigmented spots or a gray coat with obligatory white and black spots. Several independent, genetically stable germline revertants that lacked the duplicated wild-type sequence but retained the targeted codon were then derived. These animals were normally pigmented, indicating that the serine-to-alanine mutation is not deleterious to melanocyte development. The fact that mosaic coat reversions occur in all mice lacking the neo-cassette and that approximately 1% of these transmit a reverted allele to their offspring places this mutation among those with the highest spontaneous reversion rates in mammals.