Figure 3. Isolation of homozygous mutants after injection of Gb′lac2 ZFN mRNAs into crickets.

(a) An illustration of the scheme used to isolate homozygous mutations in an endogenous gene. Mutagenized G₀ adults were crossed to wild-type (WT) adults, and G₁ embryos were checked for heterozygous mutations in a first round of screening using the Surveyor nuclease. Positive G₁ individuals were developed into final-instar nymphs and subjected to a second round of screening with Surveyor nuclease assays. Positive G₁ adults were then crossed for each strain to obtain homozygous mutants in G₂ generation. (b) Germline mutations in G₀ crickets were detected in a first-round screening of G₁ embryos using Surveyor nuclease assays. The arrowhead indicates the product cleaved by the Surveyor nuclease in the f-2 line. Arrow indicates 200-bp size marker. (c,d) Isolation of heterozygous mutants from G₁ crickets in f-2 (c) and f-17 (d) lines detected in the second round of screening. Heterozygous mutant G₁ crickets were detected in a second round of screening of T3 legs from G₁ final-instar nymphs. Nymphs that harboured mutations caused by ZFNs are underlined. Arrowheads indicate the products generated from Surveyor nuclease assays. Asterisks indicate the presence of homoduplex PCR products from mutant alleles. Arrows indicate 200-bp size marker. (e) Sequence analysis of Gb′lac2 mutant alleles induced by ZFNs. Wild-type sequences are shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Boxed sequences represent ZFN-binding sites. Asterisks indicate frame-shift mutations are present. (f–h) Phenotypes of G₂ heterozygous and homozygous mutant crickets 1 day after hatching. Control first-instar nymphs were coloured black until 1 day posthatching, while heterozygous mutants were still grey in colour and homozygous mutants were white. (i) Genotyping of G₂ heterozygous and homozygous mutants. In Surveyor nuclease assays, cleavage products were detected in heterozygous mutants (indicated with an arrowhead), but not in WT or homozygous mutants (right panel). The asterisk indicates heteroduplex PCR products from WT and mutant alleles were present. Arrow indicates 200-bp size marker.