Fig. 1

Fig. 1 Generation of znf706 conditional knockout mutants by double transgenic system. A: Bar plots showing expression levels of znf706 in WT oocytes (Zhang et al., 2018), PGCs, and somatic cells in embryos (D'Orazio et al., 2021). B: ISH of znf706 showing znf706 expression patterns during oogenesis and early embryonic developmental stages. C: Left, schematic diagram of generation of Tg(piwil1:zCas9;U6:znf706 gRNAs) double transgenic system. The target sites of gRNA1 and gRNA2 at the znf706 locus were also delineated. Right, schematic diagram of generation of maternal cKO and MZ cKO mutants. D and E: Relative expression levels of zCas9 (D) and gRNA2 (E) in maternal cKO embryos derived from four Tg(piwil1:zCas9;U6:znf706 gRNAs) females detected by qRT-PCR assay. F: Relative expression levels of znf706 in WT, maternal cKO, paternal cKO, and MZ cKO embryos at 1-cell stage, detected by qRT-PCR. ns, not significant; ∗∗∗∗, P < 0.0001. G: Bar plot showing the fraction of WT embryos (WT, blue) and embryos containing mutated znf706 alleles (Mut, red) in maternal cKO and MZ cKO embryos at indicated stages. H: Sanger sequencing of znf706 alleles in WT and embryos containing mutated znf706 alleles in (G). The gRNA target site was indicated by the red dashed box. I: Examination of znf706 mutant alleles in MZ cKO embryos at 18-somite and 24 hpf by T7E1 assay. Genomic DNAs extracted from WT embryos acted as Neg. Con. FPKM, fragments per kilobase of exon per million fragments mapped; st., stage; ISH, in situ hybridization; MZ, maternal-zygotic; cKO, conditional knockout; WT, wild-type; Mut, mutant; hpf, hours post fertilization; T7E1, T7 endonuclease I; Marker, DNA markers; Neg. Con., negative control. Scales bars, 20 μm (B, upper); 100 μm (B, lower).