Generation and analysis of zebrafish with mosaic gonads: Cas9-based approach. (A) Embryos of WT or sod2 KO backgrounds homozygous for Tg(cldnB:lynEGFP) (Tg(GFP)+/+) were injected at the 8-cell stage into one of the middle blastomeres with Cas9 protein and two guide RNAs targeting egfp and sod2 sequences (sgEGFP and sgSod2). This manipulation affected only a fraction of germ cells per embryo, allowing for a high-throughput generation of animals with mosaic gonads containing WT germ cells (expressing Sod2 and EGFP transgene) and KO germs cells (where both Sod2 and EGFP functions were eliminated). Such embryos were raised to adulthood. Adult males were outcrossed to WT females, and the percentage of GFP-negative embryos (lacking EGFP and Sod2 expression) within the clutch was calculated. Results are presented in graph (B). The results of the negative control experiment with animals injected only with sgGFP (manipulating GFP expression only) are presented in graph (C). n–number of embryos and N - number of adult fish analyzed. ****p < 0.0001, n.s.p > 0.05 was determined by Student’s t-test.
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