Mouse models to investigate in situ cell fate decisions induced by TP53 and other factors (CUT&RUN)

We developed triple-FLAG TRP53 knock-in mice for TRP53 pull-down and two TRP53 response reporter mice, knocking tdTomato or GFP into the Puma/Bbc3 or p21 gene loci, respectively. By crossing the reporter mice onto a TRP53-deficient background, we could show that these reporters reliably inform on TRP53-dependent and TRP53-independent initiation of the apoptotic as well as the cell cycle arrest/senescence programs in vitro and even in vivo. FLAG-TRP53 pulldown was used for CUT&RUN analysis to identify binding of TRP53 to its target genes. Murine dermal fibroblasts (MDFs) were generated from tails of 8-12 weeks-old mice from FLAG-Trp53 KI/KI, FLAG-Trp53 KI/+, and WT (negative control) genotypes and were treated for 6h in vitro with either Nutlin-3a or DMSO (vehicle control). Cell extracts were made and subjected to pulldown using an antibody against P53-FLAG antibody.