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Abstract (1. Laboratory of Animal Nutrition and Human Health, Hunan Normal University, Changsha, 410081, China; 2. Chenzhou Science and Technology Innovation Service Center, Chenzhou 423000, China)
Abstract: The hoxc8a gene is a member of the Hox gene family, which is evolutionarily highly conserved. It has been studied that the hoxc8a gene is expressed in the swim bladder and pectoral fins of zebrafish, and that Hox genes can affect the formation and development of vertebrate body axes, whereas the mechanism of action of the hoxc8a gene is not clear. In order to study the developmental biology functions and its molecular regulation mechanism of this gene, the gene will be knocked out using CRISPR/Cas9 gene editing technology. First, target sites as well as genotype detection primers were designed on exon 1 of the hoxc8a gene, and guide DNA (sgDNA) was obtained by PCR. Then the sgDNA was recovered and purified, and the purified sgDNA was used as a template to synthesize guide RNA (sgRNA) by in vitro transcription, and finally, RNA was purified. The sgRNA and Cas9 protein were mixed in a 1:1:0.8 ratio, and the mixture was injected into wild-type zebrafish embryos using microinjection technique, and the mutant F0 generation zebrafish were screened by genotyping. When the F0 generation zebrafish were sexually mature, they were crossed with wild-type zebrafish, and the offsprings were genotyped and screened to obtain the mutant heterozygotes of the F1, and the F1 generation of zebrafish with the protein code-shifting mutation were raised after Sanger sequencing, and continued to be bred to adult. The F1 zebrafish were crossed, and the offsprings were genotyped and screened to obtain F2 homozygotes. Finally, a calcium staining test was performed on mutant homozygous and wild-type zebrafish, and it was found that the spine development of the knockout mutant was not significantly different from that of the wild-type, suggesting that the hoxc8a gene does not affect the early spine development of zebrafish, whereas whether the gene affects the development of other tissues and organs remains to be further investigated. Thus, by constructing zebrafish hoxc8a knockout lines, thus we can provide a research basis for more in-depth studies of the hoxc8a gene.
Key words: zebrafish; hoxc8a; spinal development; gene knockout line; homozygous mutant
（Acta Laser Biology Sinica, 2024, 33(6): 557-566）
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. [J]. journal1, 2024, 33(6): 1-6. |
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