Genetic Manipulation and Melanoma Induction in Mice

BRaf^CA, Tyr::CreER and Pten^lox4-5 mice were genotyped as previously described 17 (link),20 (link),24 (link). Cre-mediated conversion of BRaf^CA to BRaf^VE and the deletion of exons 4 and 5 of Pten were assessed by PCR as previously described. Topical administration of 4-hydroxytamoxifen (4-HT) was performed by preparing a 25-50mg/ml (65-130mM) solution of 4-HT (70% Z-isomer, Sigma) in DMSO and applying enough solution to wet the right ear, right flank and tail with a small paint brush on post-natal days 2, 3, and 4. For localized melanoma induction on the back skin, adult (6-8 weeks of age) mice were treated topically with 1-2 μl of 1.9mg/ml (5mM) 4-HT at 6-8 weeks of age using a similar protocol. Generalized induction in adult mice was performed by intra-peritoneal injection of 1mg of tamoxifen/40g mouse on 3 consecutive days. In this case tamoxifen was prepared as a 10mg/ml suspension in peanut oil. PD352901 was dissolved in 0.5%(w/v) Hydroxy-propyl-methylcellulose, 0.2%(v/v) Tween 80 (Sigma) and administered to mice daily by oral gavage at a dose of 12.5mg/kg. Rapamycin (LC Laboratories, Woburn, MA) was suspended in 0.5%(w/v) methylcellulose and administered to mice daily by oral gavage at a dose of 7.5mg/kg. Control animals in the melanoma prevention studies were administered with the relevant solvent. Tissues were prepared for analysis as previously described 17 (link),20 (link)