Fig. 4

Screening by Sanger sequencing of animals for the generation of the Gckr^P446L point mutation with (a) oligonucleotides (F₀ individual ssO-Gckr^P446L-54) or (b) lssDNA donors (F₀ individuals lss-Gckr^P446L-11 and lss-Gckr^P446L-10 and F₁ individual lss-Gckr^P446L-11.1f). The figure shows Sanger sequencing chromatograms of an amplicon generated with primers anchored external to the intended site of donor sequence integration as detailed in Additional file 15: Figure S14. a ssODN donors only yielded introduction of the intended silent mutations, while (b) lssDNA yielded the desired mutation in some individuals (F₀ 11 transmitting to 11.f) and only the silent mutations in others (F₀ 10). Note that founders appeared homozygous (ssO-Gckr^P446L-54, lss-Gckr^P446L-11 and lss-Gckr^P446L-10) when analyzed by Sanger sequencing, but also could contain deletion alleles in trans, as suggested by copy counting (lss-Gckr^P446L-11 in Table 4). A summary of the microinjection session outcomes is detailed in Table 3, and raw sequencing data are provided in Additional file 16