Fig. 4

Precise gene integration of a donor vector containing the GFP gene into the gap43 locus. (a) Schematic illustration of genotyping at the locus. The primer pair (GAP43-for-Seq-Fw and GAP43-for-Seq-Rv) was used for investigating the precise integration into the gap43 loci. The amplicons with or without the insert sequence are 2.0 kbp or 1.1 kbp, respectively. (b) Sequence analysis of the PCR amplicons (2.0 kbp) containing GFP gene. (Upper): Sequence observed in the F₁ fish with the inserted gene (#1–#5) (Middle): Sequence in the donor plasmid, pBaitD-gap43_500 bp-mAG (Plasmid). (Lower): Sequence in the wild type fish without the insertion (WT). (c) Electrophoresis image of the PCR products. The shorter PCR product (1.1 kbp) amplified from the intact allele was detected in both a wild type (WT) and five F₁ fish with the insertion (#1–#5). The longer PCR product (2.0 kbp) was detected only in the F₁ fish with the insertion (#1–#5)