*2.1. Embryos and Placentas Carrying KITD816V Mutation Su*ff*er from Severe Growth Retardation*

Previously, we generated the R26-LSL-KITD816V mouse line, which allows for conditional Cre-induced expression of chimeric KITD816V receptor and a GFP reporter driven by the ROSA26 promoter. The KITD816V cDNA is linked to the GFP cDNA via the coding sequence for a viral 2A peptide [27]. Here, mice carrying the ROSA26-KITD816V-GFP transgene were mated with Deleter-Cre mice, inducing ubiquitous expression of the transgenic receptor in the embryonic as well as the extra-embryonic tissue. Presence of ROSA26-KITD816V and Cre transgenes was verified by genotyping PCR using genomic DNA obtained from yolk sac or embryo (Supplementary Materials Figure S1A). Animals/placentas harboring both the Cre- and the ROSA26-KITD816V allele are further referred to as KITD816V animals/placentas. Presence of transgenes in KITD816V animals was further validated by analyzing RNA expression and protein levels in KITD816V placentas (Figure 1A–C). Of note, in KITD816V animals expressing human *KITD816V*, the expression of murine *Kit* remained unchanged, showing that endogenous *Kit* expression is not affected by transgene induction (Figure 1A,B). As expected, KIT as well as 2A-peptide proteins were present in KITD816V placentas only (Figure 1C). For further analyses, embryos and placentas were obtained on E9.5–E11.5 from KITD816V and wildtype (WT) animals. KITD816V embryos and placentas dissected on E11.5 showed growth retardation and developmental delay (Figure 1D). We previously reported that KITD816V expression restricted to the embryo proper leads to disturbance of the hematopoietic system and that such animals die at E14.5. Therefore, we hypothesize that severe growth retardation observed in KITD816V animals at 11.5 is an effect of placental insufficiency. Of note, KITD816V embryo and placentas showed GFP positivity (Figure 1D).
