FIGURE 5

FIGURE 5

Effect of lssDNA 3′ homology arm length on knock-in efficiency. (A) Donor lssDNA templates with different 3′ homology arm lengths used for comparison. (B) Schematic illustration of the sox3 knock-in allele and knock-in allele-specific PCRs for 5′ and 3′ junctions. Each lssDNA was microinjected with 1.5 fmol of the RNP complex into the cytoplasm of one-cell stage zebrafish embryos, and genomic DNA was extracted from 20-embryo pools. (C) Agarose gel image showing the PCR amplicons of knock-in allele-specific PCRs and the β-actin2 gene-specific PCR (control) to confirm DNA integrity. (D) Knock-in allele-specific qPCRs for 5′ and 3′ junctions using the hydrolysis probes shown in (B). The vertical bars represent the means of 8–10 replicates, each of which consists of a pooled sample of 10 injected embryos and is shown as a colored circle.