Journal: Wellcome Open Research
Article Title: Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo
Figure Lengend Snippet: CRISPR/Cas9 mutagenesis of lamc3 causes defects in parachordal chain (PAC) development. ( A ) Design of sgRNAs targeted to exon1 of the lamc3 gene. Each sgRNA targets a restriction endonuclease site used for genotyping and is upstream of a protospacer adjustment motif (PAM). ( B – D ) Cas9/sgRNA injected embryos (as labelled) at 50 hpf. ( B – D ) Brightfield images show no developmental defects or developmental delay. ( B′ – D′ ) Whole embryo images of Tg( fli1a :egfp) fluorescence area indicated by white dotted line is enlarged in ( B′′ – D′′ ) the PAC in each hemisegment is indicated with a red asterisk. ( E ) Quantification of Tg( fli1a :egfp) embryos at 50 hpf with PAC defects. P-values determined by Fisher’s exact Two-tailed test compared to Cas9 alone controls. ( F – I ) 2% agarose genotyping gels with 100 bp ladder. ( F ) Schematic diagram of the region amplified by PCR contains two SphI sites (orange), which would digest into three fragments of 83, 164 and 263 bp in length. Predicted mutation of one of these sites will produce two bands of 83 and 427 bp in length. A natural variant contains a SNP in the second SphI site, complete digestion of this variant would produce two bands of 164 and 346 bp in length. Mutation of the target SphI site would prevent digestion of the fragment. Digestion of the amplicon with Taq α I (blue) in wild type embryos will yield two fragments of 204 and 306 bp in size. ( G ) Restriction endonuclease digest of target region shows undigested mutant products (black asterisk) at a size of 427/510 bp in sgRNA1-injected embryos. ( H ) Fewer undigested mutant products (510 bp) were observed in sgRNA2-injected embryos. ( I ) Brightfield images sgRNA1/Cas9 injected embryo at 5 dpf shows severe oedema. ( J ) Genotyping of 3-month adult fish. A, anterior; b/bp, base pairs; d, days post fertilisation; D, dorsal; kb, kilobases; mpf, months post fertilisation; n, number of embryos; PAC, parachordal chain; PAM, protospacer adjustment motif; SBMO, splice-blocking morpholino; sgRNA, short guide RNA; U, undigested control;.
Article Snippet: Frozen embryos were thawed and homogenised in 100 μl of protein extraction buffer (1% IGEPAL, 150 mM NaCl, 20 mM Tris pH 7.5, 2 mM EDTA, 50 mM NaF, 1mM sodium pyrophosphate) with 1× cOmplete Mini Protease Inhibitor Cocktail (1183615300, Roche) overnight at 4°C with rotation.
Techniques: CRISPR, Mutagenesis, Injection, Fluorescence, Two Tailed Test, Amplification, Polymerase Chain Reaction, Variant Assay, Fluorescence In Situ Hybridization, Blocking Assay