Figure 2
In vivo functional validation of the FSFTGFβCA allele. (a) Breeding strategy ([Act-Flpe] x [FSFTGFβCA]) to generate [Act-Flpe; FSFTGFβCA] individuals. Primers (p) used for DNA genotyping (panel d) and RT-PCR (panel e) are represented by grey arrowheads. (b) [FSFTGFβCA] were crossed with [Act-Flpe] mice. Total numbers (black) and expected numbers (grey) of litters, pups, and offspring genotype distribution are presented. A χ² test was performed to statistically confirm the “loss” of a significant proportion of [Act-Flpe; FSFTGFβCA] individuals after birth. The Chi2 were calculated using Excel workbook developed by Montoliu36. (c) Kaplan Meyer disease free survival curve of mice of indicated genotypes. n represents the number of animals for each genotype. (d) PCR on genomic DNA prepared from tail snips of indicated genotypes to detect the ROSA26, Act-Flpe, the unrecombined FSFTGFβCA, and the recombined FSFTGFβCA alleles. (e) Quantification of TGFβCA (left panel) and eYFP (right panel) mRNA by RT-PCR on total RNA prepared from [FSFTGFβCA] and [Act-Flpe; FSFTGFβCA] ear skin samples. (f) Western blot analysis of eYFP and β-tubulin on whole protein extracts prepared from skin samples of indicated genotypes. In e and f, one representative experiment performed 3 times with skin samples from different mice is presented. In c and e, Prism 7.0 (Graphpad) was used to create graphs. In e, the error bars represent the standard deviation from technical duplicates.
