Figure 4.

NF1 23kb excision and analysis of mosaicism. (A) Schematic depicts alterations between NF1 exons 15/16 and 29/30, resulting in a 23kb excision. Red arrowheads: Cas9 cut sites, single-headed black arrows (f, r1, r2): primer annealing sites. (B) Competitive PCR for excision and non-excision alleles yields amplicon products of two different sizes, indicated by black arrowheads on right (r1 and r2). Included are an unedited control, a negative control (NC; a founder (G401) from another unrelated NF1 model, two 23kb excision founders (F0; blue and red labels), F1 offsprings (non-mosaic controls) and no template control (NTC). Black arrowhead (on left): 500bp, 400bp and 100bp markers on DNA ladder (NEB, N0551S). (C) Quantification of the pixel density of PCR products in (B) and additional bands not depicted in panel B (see Supplementary Figure 1 for complete gel electrophoresis image), with bars aligned with corresponding PCR gel wells to identify the most effective breeders carrying the 23kb excision. (D) Proportionality of F1 pigs from two specific founders in (B) and (C) carrying the excision allele compared to those not carrying the excision allele. Thirty-nine percent of F1 pigs produced by founder pig G1283 carried an excision allele, while none of the F1 pigs produced by founder pig G1290 did so. There was a significant association between excision allele abundance (pixel density) and germline transmission rate (logistic regression, p<0.05).