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

Figure 3. Generation of geno-tagging alleles by improving the dual-cassette donor strategy at the zebrafish tbx5a locus.

(A) Schematic diagram of the KI strategy for geno-tagging based on the tbx5a-T2A-tdT-2PA floxP tdG-2PA donor (or abbreviated as tbx5a PoR-NeG donor). Primers T5qF and T5qR are used for qRT-PCR in Figure 3—figure supplement 1E and F. (B) Results of junction PCR and direct sequencing to detect the tbx5a geno-tagging donor knockin and Cre-induced recombination events in 48 hpf embryos obtained from the cross in Figure 3—figure supplement 1B, that is the F1 embryos from the #42 positive F0 outcrossed with a wild-type zebrafish before or after Cre mRNA injection. Note that the sequences of both the T5F2 and T5R1 primers are also present in the donor vector, flanking the upstream loxP site (as shown in panel A); therefore, a 730 bp product could be amplified in the lane with the donor as the template. F1-R: an F1 embryo showing a red fluorescent signal (before Cre mRNA injection). F1-G: an F1 embryo showing a green fluorescent signal (after Cre mRNA injection). Donor: tbx5a PoR-NeG geno-tagging donor plasmid. WT: pooled genomic DNA of five wild-type embryos. (C) Phenotype analysis of the 72 hpf embryos from tbx5a+/PoR-NeG heterozygotes (derived from F0 #42 in Supplementary file 5) crossed with a tbx5a+/PoR-Ne heterozygote (derived from F0 #12 in Supplementary file 3) after the injection of Cre mRNA at the 1 cell stage. The upper panel represents an uninjected control embryo showing only a red fluorescent signal, whose genotype should be either tbx5a+/PoR-Ne, tbx5a+/PoR-NeG or tbx5a PoR-Ne/PoR-NeG. The middle panel represents a Cre mRNA-injected embryo showing normal development, whose genotype is expected to be tbx5a+/NeG. The lower panel represents a Cre mRNA-injected embryo showing a typical tbx5a mutant phenotype, including heart region defects and a lack of pectoral fins, whose genotype is expected to be tbx5aNe/NeG. Arrows indicate the pectoral fins, and arrowheads indicate the heart region. The boxed insets show a higher magnification of the corresponding heart region, for better comparison of heart morphology. Scale bar, 200 μm. (D) Z-stack confocal images of two representative 48 hpf embryos after the injection of Cre mRNA at the 2- to 8 cell stage from the same cross as in C. The white arrows indicate the colocalization of the tdGFP and tdTomato signals, and the white arrowheads indicate the mutually exclusive expression of the tdGFP and tdTomato signals. Scale bar, 50 μm.

Figure 3.

Figure 3—figure supplement 1. Evaluation of the tbx5a geno-tagging effect.

Figure 3—figure supplement 1.

(A) Preselection of tbx5a geno-tagging F0 individual by junction PCR analysis. 5’ or 3’ junctions were amplified by PCR using genomic DNA extracted from fin clips of the #1, #2, #9, #11, #24 and #42 F0 adult fish. The corresponding primer pairs are shown on the left side of the gel images, and the positions of these primers can be found in Figure 3A. (B) Switching of fluorescent signals achieved from the tbx5a geno-tagging allele after Cre mRNA injection into the F1 progeny from #42 positive F0 outcrossed with a wild-type zebrafish. The arrowheads indicate pectoral fins. The outlined boxed areas indicate the heart region, showing the change in the fluorescent signals in the heart before and after Cre mRNA injection. Scale bar, 200 μm. (C) The experimental design for the functionality test of the tbx5a geno-tagging allele. The progeny from the cross of a tbx5a+/PoR-NeG heterozygote with a tbx5a+/PoR-Ne heterozygote were divided into three groups: Group I was injected with 100 pg Cre mRNA at the one-cell stage, Group II was injected with 25 pg Cre mRNA in a single cell at the 4 cell stage, and Group III remained untreated as a control. The histogram shows the ratio of defective embryos after Cre mRNA injection in different groups. (D) Confocal images of the heart regions of two embryos from the cross of Tg(cmlc2:zCreERT2-2A-ECFP) transgenic fish with tbx5a PoR-NeG/PoR-NeG after 4-HT treatment, showing a red to green change in the fluorescent signals upon Cre induction. -S: Single-plane view, -M: Maximum intensity projection view of z-stack images. Scale bar, 50 μm. (E) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a+/NeG and tbx5a+/PoR-NeG embryos were obtained from crosses of tbx5aPoR-NeG/PoR-NeG 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. (F) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a+/NeG and tbx5aNeG/NeG embryos derived from the Cre mRNA-injected tbx5a+/PoR-NeG and tbx5aPoR-NeG/PoR-NeG embryos, respectively. The original embryos were obtained from the crossing of tbx5aPoR-NeG/PoR-NeG homozygotes with tbx5a+/PoR-NeG 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.