Fig. 4

Fig. 4 Fluorescent signals can be dramatically amplified by the VH strategy.

a mRNA levels of fluorescence-labeled connexins in F₃ zebrafish. Thirty connexin^+tag/+tag or connexin^+/+tag zebrafish larvae were pooled to extract RNA for qPCR. qPCR primers were designed to target the fluorescence tag. For the group with eGFP-labeling, data were normalized to actb1 and relative to cx43^+eGFP/+eGFP. For the mCherry group, data were normalized to actb1 and relative to cx43.4^{+mCherry/+mCherry}. Data are presented as mean ± SD of 3 independent experiments. Three repeats were included per group. P-values were calculated using an unpaired Student’s t-test. ****P < 0.0001. b Schematic diagrams of the genome after editing by direct fluorescence-labeling (DFL), V, VH, and SPH strategies, respectively. All strategies are built on the S-NGG-25 KI method. Theoretically, for the DFL strategy, eGFP is directly linked to the target gene by a P2A linker or a GSSSS₃-linker and would be co-expressed with the target gene. For the V or VH strategy, Gal4-VP64 or Gal4-VP64-HSF1 would be co-expressed with the target gene, be separated from the target protein due to the function of a P2A peptide, and then induce eGFP expression by binding 5×nrUAS. For the SPH strategy, Gal4-GCN4 would be co-expressed with the target gene, be separated from the target protein, and then work together with scFV-GCN4-P65-HSF1, which is constitutively expressed in all cells, to transcriptionally activate expression of eGFP. c Fluorescence images of F₁ embryos carrying fluorescence-labeled alleles generated by using DFL, V, VH, or SPH strategy at the cx43.4 loci at 32 hpf. Desired F₁ were screened out based on GFP expression patterns and genotyping results. Images were representatives of at least 10 desired F₁ and were captured under the same exposure time (200 ms). Scale bars, 2 mm. d GFP expression patterns in F₁ larvae generated by DFL, V, VH, or SPH strategy at the cx43.4 locus. GFP was detected in the entire notochord of F₁ generated by DFL or VH but was only partially in the notochord of F₁ generated by V or SPH at 48 hpf. Exposure time was determined by the fluorescence intensity of GFP signals. Scale bars, 100 µm. Images are representatives of at least 10 F₁ larvae. e Images of F₁ larvae for cx34.4 or cx52.6 KI generated by a DFL strategy using a P2A linker or a VH strategy. GFP signals were detected in the spinal cord and hindbrain at 48 hpf in cx34.4^+/+VH embryos and in the forebrain, midbrain, and posterior notochord at 48 hpf in cx52.6^+/+VH embryos. Scale bars, 100 µm. Images are representatives of at least 10 F₁ larvae.