Transcriptional Response to transgenic BRAF(V600K) in the intestinal epithelium

We have analysed transcriptional effects of BRAF(V600K) in the intestinal epithelium of transgenic mice that harbour an inducible BRAF(V600K) transgene. Mice used in this experiment were compound transgenic for a stem-cell specific Lgr5-EGFP Reporter and either an inducible TdTomato-2A-BRAF(V600K) transgene or an inducibe TdTomato-luciferase transgene. Transgenes were induced by doxycycline (4mg/ml) treatment provided in a 1% sucrose solution in the drinking water of transgenic mice for 24h. Intestinal crypts were isolated by filtering (70um) following a PBS/EDTA incubation step. Single cell suspensions were made by digestion of crypts in 500ug/ml trypsine and 0.8u/ml DNAseI for approx. 20min at RT. Cell populations for profiling were isolated by FACS, using an Aria SOPR (BD), equipped with a 70um nozzle.