Fig. 5.

Selection of biallelic mutants using two different colors of fluorescence. (a) Fluorescence of F₂ embryos derived from mating with each F₁ monoallelic mutants harboring either the GFP or the RFP gene in the gap43 locus. Each of the F₂ embryos were categorized into the following four groups; no fluorescence (G−/R–), green fluorescence (G+/R–), red fluorescence (G−/R+), and both green and red fluorescence (G+/R+). The images were captured at 4 days post fertilization (dpf). White arrowheads show fluorescence in the central nervous system (CNS), while white arrows show autofluorescence of bacteria on the surface of the egg membrane. (b, c) PCR genotyping of the F₂ embryos derived from mating between the F₁ monoallelic mutants. (b) The EF1-α fragment (519 bp) was used to confirm genomic DNA extraction, and was detected in all samples. The gap43 fragment (551 bp) was detected from the groups (G−/R–), (G+/R–), and (G−/R+). The GFP fragment (749 bp) was detected from (G+/R–) and (G+/R+) groups, while the RFP fragment (698 bp) was detected from (G−/R+) and (G+/R+) groups. In (G+/R+), no PCR product of gap43 was detected. (c) Design of PCR primers to detect the targeted gene integration into the gap43 locus. (Upper): Genomic structure of the intact gap43 locus. The size of the PCR product amplified using primer pair (GAP43-for-Seq-Fw and GAP43-Rv) is 551 bp. The latter primer is 20 bp in size and is shown as a red arrow. The reverse primer is designed on the boundary between upstream and downstream long homology arms, so that this primer pairs can amplify the intact locus but not the GFP- or RFP-integrated loci. (Middle): Genomic structure of gap43 locus at which the donor plasmid harboring the GFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and mAG-Rv) is 749 bp. (Lower): Genomic structure of the gap43 gene at which the donor plasmid harboring the RFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and tdTomato-Rv) is 698 bp