FIGURE 6

FIGURE 6

Founder germ cell mosaicism. (A) Mosaicism analysis by PCR using genomic DNA from F1 embryo pools. F1 embryos from out-crosses of the founders with wild-type fish were pooled (50–100 embryos per pool) and used for genomic DNA preparation. The PCR primers bind outside of the homology arms to amplify both wild-type (WT) and knock-in (KI) alleles as indicated by the arrows. The agarose gel electrophoresis image represents the PCR amplicons of each founder along with the wild-type fish. (B) Mosaicism analysis by PCR using genomic DNA from individual F1 embryos. Possible genotypes of germ cells of biallelic knock-in founders and F1 embryos are illustrated. Individual F1 progeny embryos from out-crosses of FLAGx3-50_#16 (a) and PAx3-50_#21 (b) founders were subjected to genomic DNA preparation and PCR amplification of 5′ and 3′ junctions of the composite tag-modified sox3 gene. A total of 20 embryos were analyzed per founder. The number of PCR positive embryos per total embryos for each PCR amplification is shown on the right side of each agarose gel image. The minor knock-in allele with imprecisions is indicated with green arrows (Ba).