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

Figure 2. The tbx5a and kctd10 conditional alleles are responsive to Cre recombinase.

(A) Images of the 72 hpf progeny (F1) from a tbx5a+/Δ5 heterozygote crossed with the F0 #12 (mosaic for the tbx5aPoR-Ne allele) against the Tg(cmlc2:EGFP) transgenic background with or without Cre mRNA injection. The fluorescent images were obtained in the lateral view. Black arrowheads indicate pectoral fins, and black or white arrows indicate the heart. A: atrium. V: ventricle. Scale bar, 200 μm. (B) Genotyping results of the individual Cre mRNA-injected embryos obtained from the cross in A. (C) Images of the 48 hpf progeny (F2) derived from the cross of two kctd10 KI heterozygotes (F1), each carrying a different KI allele (kctd10PoG-Ne-1 from #32 and kctd10PoG-Ne-2 from #5) with the Tg(cmlc2:EGFP) transgenic background, to reveal the morphology of the heart. The white dotted line indicates the outline of the heart. The hearts in the upper and middle panels developed normally, showing obvious heart looping. In contrast, the heart in the lower panel shows defective development, exhibiting AVC malformations and heart looping failure. Black arrowheads indicate the heart. Scale bar, 200 μm. (D) Genotyping by PCR amplification of the region flanking the loxP recombination site of the Cre mRNA-injected individual embryos obtained from the cross in C. (E) Representative junction PCR and direct sequencing results of the Cre mRNA-injected individual embryos showing normal or defective heart development obtained from the cross in C. As expected, the results indicate that the embryo showing the heart phenotype (labeled as ‘Single defective embryo’ in the figure) was a kctd10PoG-Ne-1/PoG-Ne-2 compound heterozygote (F2) before Cre mRNA injection since it showed overlapping peaks (red boxed region) in the sequencing results of the PCR products at both the 5’ and 3’ junctions (right panel), as the two alleles have different indel sequences at the junction sites. In contrast, the normal embryos (labeled as ‘Single normal embryos’ in the figure) were either kctd10+/PoG-Ne-1 or kctd10+/PoG-Ne-2 heterozygotes before Cre mRNA injection and therefore displayed uniform sequencing results corresponding to either the kctd10PoG-Ne-1 (or kctd10Ne-1) or kctd10PoG-Ne-2 (or kctd10Ne-2) allele, respectively. The expected corresponding sequences can be found in Figure 2—figure supplement 2J and K.

Figure 2.

Figure 2—figure supplement 1. Evaluation of the indel efficiency of the tbx5a E3 target site and phenotype analysis of the tbx5a indel mutation.

Figure 2—figure supplement 1.

(A) The position and sequence of the tbx5a exon 3 (E3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Targeting efficiency evaluated by PCR and AluI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 90%. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Approximately 25% of embryos from the incross of tbx5a+/Δ5 heterozygotes showed defects in heart (black arrows) and pectoral fins (black arrowheads). Genotyping results revealed that all the defective embryos were tbx5aΔ5/Δ5 homozygotes (lower panel), while the siblings showed a normal morphology. The Tg(cmlc2:EGFP) transgenic background was introduced to reveal the heart morphology, and all the defective embryos also showed failure of cardiac looping. The dotted lines denote the outline of the heart. Scale bar, 200 μm. (E) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-Ne donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a+/Ne and tbx5a+/PoR-Ne embryos were obtained from the crossing of the tbx5a PoR-Ne/PoR-Ne homozygotes with wild-type zebrafish with or without injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. (F) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a+/Ne and tbx5aNe/Ne embryos derived from the Cre mRNA-injected tbx5a+/PoR-Ne and tbx5aPoR-Ne/PoR-Ne embryos, respectively. The original embryos were obtained from the crossing of tbx5aPoR-Ne/PoR-Ne homozygotes with tbx5a+/PoR-Ne heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t-test was applied to calculate p values in all the experiments. *: p<0.05. ***: p<0.001. NS: Not significant.
Figure 2—figure supplement 2. Strategy and evaluation of the targeted insertion of the PoG-Ne donor at the kctd10 locus.

Figure 2—figure supplement 2.

(A) The position and sequence of the kctd10 intron 1 (I1) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Targeting efficiency evaluated by PCR and Hpy188I restriction endonuclease digestion. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Schematic diagram of the kctd10-2A-td GFP floxP 2PA-mutExon PoNe donor (abbreviated as kctd10 PoG-Ne donor) and the strategy of targeted insertion and conditional knockout using the CRISPR/Cas system. Primers K10qF and K10qR are used for qRT-PCR in L and M. (E) Images of a 10 hpf F0 zebrafish embryo after the injection of the kctd10 PoG-Ne donor together with zCas9 mRNA and corresponding gRNAs. White arrows indicate tdGFP signals. Scale bar, 200 μm. (F) Junction PCR to detect NHEJ-mediated knockin events in the injected founder embryos. Injected: Donor+Cas9/gRNA-injected embryos. Donor: kctd10 PoG-Ne donor plasmid. Uninjected: Uninjected embryos. (G) Images of a 10 hpf F1 zebrafish embryo from an outcross of the kctd10 PoG-Ne donor KI-positive F0 female (#32) shown in Supplementary file 4, bearing the kctd10PoG-Ne-1 allele. Strong maternal expression of tdGFP can be clearly observed in this F1 embryo. Scale bar, 200 μm. (H) Schematic diagram of the kctd10 KI allele, showing the position of the primers used for junction PCR in I-K and qRT-PCR in L. A new primer pair was used to amplify the 3’ junction of the F1 embryos. (I) Junction PCR to detect the knockin allele in individual F1 embryos (1-4) from the cross in G. Note that not all of the embryos inherited the knockin allele from the F0 female, indicating germline mosaicism of this adult fish. (J) Sequencing results of the PCR products from the two positive embryos (2 and 3) in I, which showed the same junction sequence of the kctd10PoG-Ne-1 allele. (K) Sequencing results of the PCR products (using the same primer pair as in I and J) from an EGFP-positive F1 zebrafish embryo obtained from an outcross of the positive F0 male (#5), representing the junction sequence of the kctd10PoG-Ne-2 allele. (L) qRT-PCR results showing the transcription level of the kctd10 locus in wild-type (WT) and kctd10 PoG-Ne donor KI zebrafish embryos at 72 hpf, using K10qF and K10qR primers. The kctd10+/Ne-1 and kctd10+/PoG-Ne-1 embryos were obtained from the cross of kctd10 PoG-Ne-1/PoG-Ne-1 homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. (M) qRT-PCR results using K10qF and K10qR primers, showing the transcription level of the kctd10 locus in the kctd10+/Ne-1 and kctd10Ne-1/Ne-1 embryos derived from the Cre mRNA-injected kctd10+/PoG-Ne-1 and kctd10PoG-Ne-1/PoG-Ne-1 embryos, respectively. The original embryos were obtained from the crossing of kctd10PoG-Ne-1/PoG-Ne-1 homozygotes with kctd10+/PoG-Ne-1 heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t-test was applied to calculate p values in all the experiments. *: p<0.05. **: p<0.01. ***: p<0.001. NS: Not significant.