Fig. 3: Site-specific integration of targeting vector into tdgf1.
From: CRIMP: a CRISPR/Cas9 insertional mutagenesis protocol and toolkit
a Schematic of tdgf1 gene structure and location of the intron 3 guideRNA target site. b The pSA2-Gal4vp16/4xnrUAS-mTagBFP2 targeting vector, FRT and FRT3 sites are indicated by triangles. c Successful integration of the targeting plasmid to generate the Ti(tdgf1int3-Gal4vp16/4xnrUAS-mTagBFP2) targeted insertion line. d Brightfield and fluorescent images of Ti(tdgf1int3-Gal4vp16/4xnrUAS-mTagBFP2) targeted insertion line demonstrating loss of tdgf1 phenotypes in homozygote mutants at 24 hpf. mTagBFP2 fluorescence is false-coloured magenta. Scale bars = 250 μm. e, f mRNA expression analysis at 24 hpf demonstrates loss of tdgf1 in homozygote mutants (e) and the fold increase of mTagBFP2 expression levels compared to native tdgf1 from Gal4/UAS amplification (f). Error bars represent SEM for n = 3 independent biological replicates, each consisting of a pooled sample of 14 or 16 embryos. rpl13 and ef1α were used as the reference genes. Source data are provided as a Source Data file. Statistical differences were determined using a two-tailed unpaired t-test.
