Fig. 1

Generation and validation of the NTN1-T2A-GFP chicken line. (a) Expression of endogenous NTN1 in wild type chicken embryo in somite regions (between asterisks), neural tube (arrowheads), and in pioneer cell regions at the fissure edges (arrow, inset), and fluorescent in situ hybridisation for NTN1 mRNA (green, arrowheads. Scale bar, 50 μm. Blue = DAPI; Magenta = Laminin. (b) Schema of genome editing strategy with sgRNA ___location (red arrow) shown in genome and vector. Abbreviations: EGF, Epidermal growth factor ___domain; NTR, N-terminal region; PAM, protospacer motif. Blue arrows indicate locations of diagnostic PCR primers. (c) Strategy for generating germ-line gene edited NTN1-T2A-eGFP chicken line via transfection of PGCs and injection of PGC clones into surrogate host embryos. (d) Diagnostic PCRs indicate monoallelic (NTN1^GFP/WT) or biallelic (NTN1^GFP/GFP) HDR-mediated integration of RTs in clonal PGCs (higher molecular weight band). (e) DNA sequence trace and corresponding amino-acid translation showing successful in-frame HDR in clonally propagated PGCs. Arrow indicates predicted T2A self-cleavage site. (f) RT-PCR for transgene expression in neural tube (NT) and optic fissure (OF) dissected from WT or NTN1^{NTN1 − T2A−GFP/+} embryos. Crops from separate agarose gels are displayed. (g) Whole NTN1^{NTN1 − T2A−GFP/+} embryo with GFP expression detected in neural tube (arrowheads), optic fissure (arrows), and somite regions (asterisks). (h) Flat-mount NTN1^{NTN1 − T2A−GFP/+} HH28/E6 dissected neural tube (Left) opened by cutting dorsally with GFP expression (arrowheads) observed in the floor-plate region, and ventral retina (Right) with GFP detected at the edges of the fissure margin (arrowheads). Full uncropped agarose gels are shown in Supplemental Fig. 1.