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. 2019 Oct 30;8:e48081. doi: 10.7554/eLife.48081

Figure 1. Generation of CKO coupled with gene labeling dual-function alleles through targeted insertion in zebrafish at the tbx5a locus.

(A) Schematic diagram of the KI strategy based on the dual-cassette PoNe donor tbx5a-T2A-tdTomato floxP 2PA-mutExon (the tbx5a PoR-Ne donor), consisting of a Po-cassette and a Ne-cassette (highlighted by pink and yellow shadows, respectively). The target sequences of hEMX1 and tbx5a are shown in purple and brown, respectively, and the PAMs are in shown green. Black triangles represent loxP. Black and gray diamonds indicate polyadenylation (PA) signals. The black bar in the third exon (E3) indicates the in-frame premature termination codon (PTC). Primers T5qF and T5qR are used for qRT-PCR in Figure 2—figure supplement 1E and F. (B) Images of F1 larvae from an outcross of a tbx5a PoR-Ne donor knockin founder (#12), showing tdTomato expression in the pectoral fins (white arrows), heart (white arrowheads), eyes (white dotted circle) and nervous system (white asterisk). Scale bar, 200 μm. (C) Images of F1 progeny from Tg(cmlc2:EGFP) transgenic zebrafish crossed with the tbx5a KI founder (#12), showing an antero-posterior gradient of tbx5a expression in the ventricle. Upper panel: Ventral view of a 72 hpf embryo. Lower panel: Z-stack confocal images of the heart region from a 48 hpf embryo. A: atrium. AVC: atrioventricular canal. OFT: outflow track. V: ventricle. Scale bar, 50 μm. (D) Junction PCR and direct sequencing results of individual positive F1 progeny from outcrosses of each of the three positive founders (#3, #5 and #12). Due to an extra copy of the T5R1 primer sequence in the PoR-Ne donor, PCR with the primer pair T5F2 and T5R1 targeting the donor plasmid results in a larger fragment than that of the F1 progeny. F1: an F1 embryo from F0 #12. Donor: tbx5a PoR-Ne donor plasmid. WT: pooled genomic DNA of five wild-type embryos.

Figure 1.

Figure 1—figure supplement 1. Evaluation of the expression of tbx5a and targeted insertion of the CKO + gene labeling PoNe donor at the tbx5a I2 target site in founder embryos.

Figure 1—figure supplement 1.

(A) The position and sequence of the tbx5a intron 2 (I2) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Indel efficiency evaluated by PCR and BtgI restriction endonuclease digestion. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Mosaic expression of tdTomato in the heart (white arrowhead) and fin (white arrows) in a founder embryo after the injection of gRNAs purified by LiCl precipitation together with zCas9 mRNA and the tbx5a PoR-Ne donor from Figure 1A. Scale bar, 200 μm. (E) Junction PCR to detect NHEJ-mediated knockin events in founder embryos. The expected products (870 bp and 570 bp) were obtained by amplification with the corresponding primers shown in Figure 1A. Injected: Donor+Cas9/gRNA-injected embryos. Donor: tbx5a PoR-Ne donor plasmid. Uninjected: Uninjected embryos. (F) The expression of tbx5a in the zebrafish heart shown by whole-mount in situ hybridization (ventral view). The dotted lines denote the outline of the heart. Scale bar, 100 μm.