Generation of a mouse model to study the temporal relationship between BRAFV600E expression and PTEN silencing in melanoma development

Metastatic melanoma is a devastating and poorly understood disease that is extremely resistant to current approved therapeutic regimens. The earliest and most common observed genetic alteration encodes constitutively active BRAF-V600E (BRAFV600E) which promotes sustained activation of the BRAF-MEK-ERK MAP kinase signaling pathway. BRAFV600E is detected in 85% of human nevi and ~65% of metastatic melanomas. Progression from nevi to melanoma is thought to require the subsequent functional loss of at least one tumor suppressor gene, commonly PTEN. We have previously demonstrated that the concomitant activation of BRAFV600E and PTEN silencing led to the appearance of highly pigmented melanocytic skin lesions with histological features of melanoma. However, human tumors do not develop mutations simultaneously. Thus, to study melanoma progression in vivo, we will initiate BRAFV600E expression and PTEN silencing independently with Flp and Cre respectively. We have previously generated mice carrying a Flp-activated BRAFV600E allele (BRAFFA), which expresses normal BRAF prior to Flp-mediated recombination, at which point BRAFV600E is expressed. Similarly, we have a conditional PTEN allele that becomes inactivated upon Cre-mediated recombination.To separate BRAFV600E activation and PTEN silencing in vivo, I attempted to generate a transgenic mouse expressing inducible forms of Cre and Flp recombinases in a melanocyte-specific manner (pTyr:CreER and pTyr:FlpPR). The two constructs were co-integrated into the genome of C57BL/6 zygotes, generating five Tyr:FC founder strains. In vitro analysis of the constructs demonstrated melanocyte specificity and appropriate inducibility and specificity for recombination sequences. However, zero of five Tyr:FC strains demonstrated recombinase expression or function in vivo. The reason behind this lack of expression remains unknown but is most likely attributed to the co-injection technique, too few founder strains for analysis, or faults in the pTyr constructs.