Supplementary Figure 4: Genomic PCR and Sanger sequence analysis of individual F0 Pax7:Cherry knock-in axolotls.
(a) Genomic PCR of the Pax7 locus of individual F0 Pax7:Cherry knock-in axolotls (animal #1–4) and the negative control (Ctr, un-injected sibling of the F0 knock-in axolotls). Upon NHEJ-mediated insertion of the Cherry reporter gene into the Pax7 locus, approximately 2.8 kb PCR products are amplified using the primer pair Pax7-fw and pA-rev (Fig. 7 and Table 4). (b) Sequence analysis reveals that in-frame scars (5’ integration junctions, animal #1: 15nt deletion; animal #2: 9nt deletion; animal #3: 9nt and 7nt deletions; animal #4: 6nt deletion) are present in all F0 Pax7:Cherry animals. Note: the in-frame integration junctions in the limb (limb junct) and tail (tail junct) from the same individuals are identical in three different knock-in axolotls, animal #1, #2 and #4. All animal experiments were carried out according to relevant Institutional and National regulations. Adapted with permission from Fei, J.F. et al. Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. Proc. Natl. Acad. Sci. USA 114, 12501–12506 (2017).
