Mitf over-expression leads to microphthalmia and coloboma in Mitf-cre mice
The Microphthalmia associated transcription factor (Mitf) is a critical regulator of the melanocyte lineage and also plays an important role in eye development. Mitf activity in different cell types is controlled in part by ten alternative promoters and their resulting isoforms. A useful tool for melanocyte-based research, the Mitf-cre transgene was designed to express Cre recombinase from the Mitf-M promoter, which is melanocyte specific. However, Mitf-cre mice are also microphthalmic, perhaps because of insertional mutagenesis or disrupted gene expression. Here, we investigated these possibilities. We determined that the eye phenotype arises early, with Mitf-cre embryos at E13.5 exhibiting variable ocular sizes and abnormalities, but all with coloboma. Targeted locus amplification and next generation sequencing indicated that multiple copies of the transgene integrated into an intergenic region on chromosome 2, in between Spred1 and Meis2. The BAC transgene used to make Mitf-cre was larger than expected, carrying three upstream alternative promoters, Mitf-H, Mitf-D, and Mitf-B, which could express their isoforms intact off the transgene. RT-qPCR using eye tissue demonstrated a 5-fold increase in Mitf transcripts containing exon 1B1b, which is shared by Mitf-H, Mitf-D, and Mitf-B, while Spred1 and Meis2 did not differ in their expression. These findings clarify and support the usage of Mitf-cre in conditional mutagenesis in melanocytes. The specific over-expression of the Mitf-H and Mitf-D isoforms, which are preferentially expressed in the RPE, presents a unique resource for those interested in eye development and coloboma.