Fertility and embryonic development of the established paqr5b?/? line. (A) DNA sequence and predicted protein structure of an established paqr5b gene-edited strain. (Uppest panel) DNA sequences around the target site (in red) for CRISPR/Cas9 digestion in wild-type (paqr5b+/+) and paqr5b?/? mutants are indicated. A 13-nucleotide deletion was induced in the target site in the selected mutant. (Middle panel) The predicted protein sequences of the paqr5b+/+ and paqr5b?/? mutants are indicated. It was expected that a peptide of 74 amino acids in length with an N-terminal 18 amino acid sequence produced in the paqr5b?/? mutant was the same as that produced in wild-type Paqr5b zebrafish. Thus, only an N-terminal short fragment without a transmembrane region is present in the Paqr5b protein produced in the paqr5b?/? mutant. (Lowest panel) Diagrams of the predicted protein structures of the paqr5b+/+ and paqr5b?/? mutant strains are shown. The predicted transmembrane regions and the altered amino acid regions in the paqr5b?/? mutant are indicated by black boxes and diagonal boxes, respectively. (B) The relative expression levels of paqr5b in the olfactory rosette (OR) of the wild-type and the paqr5b mutants were compared. mRNA abundance was measured in triplicate for each sample from 10 fish, and all data from three preparations were normalized by the number of elongation factor 1? (EF1?) transcripts in each sample. The data indicated the expression level of the wild type set as 100. (C) Fecundity of paqr5b+/+ and paqr5b?/? zebrafish. The total number of eggs crossing each line was counted (n = 4, ** P ? 0.001). (D) Representative photograph of developing embryos with different sizes of chorions. The scale bar is 1 mm. Comparison of choroidal diameter between paqr5b+/+ and paqr5b?/? embryos (n = 4, **** P ? 0.00001). (E) Representative photograph of embryos at 5 dpf. Typical head abnormalities and spine curvatures in the paqr5b?/? embryos are indicated by arrows. The scale bars in the upper panels are 1 mm. The scale bars in the lower panels are 500 ?m. (F) Comparison of the percentage of abnormal embryos between the paqr5b+/+ and paqr5b?/? embryos (n = 4, **** P ? 0.00001). (G) Comparison of brain transverse sections of paqr5b+/+ and paqr5b?/? embryos. The ventricle of the embryo brain is indicated by arrows. The scale bars are 20 ?m.
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