This thesis firstly summarized the evolutional process of immune system, advantages and advance in hematopoiesis by using zebrafish as model, the significance of T regulatory cell in immunity and the regulation of T regulatory cell by foxp3 gene. Referring to CRISPR-Cas9 technique protocol, the author selectively designed 4 sgRNAs, targeting the N-terminal repression domain and C-terminal fork-head domain of Foxp3a protein in the gene exons. By constructing sgRNA and Cas9 protein expression vectors, which were then transcribed in vitro to obtain sgRNA and Cas9 mRNA, the author microinjected Cas9 mRNA mixed with sgRNA into single cell embryos. Then, using polymerase chain reaction, enzyme digestion and other common techniques to identify sgRNA with mutation efficiency and chimaera...[ Read more ]

This thesis firstly summarized the evolutional process of immune system, advantages and advance in hematopoiesis by using zebrafish as model, the significance of T regulatory cell in immunity and the regulation of T regulatory cell by foxp3 gene. Referring to CRISPR-Cas9 technique protocol, the author selectively designed 4 sgRNAs, targeting the N-terminal repression domain and C-terminal fork-head domain of Foxp3a protein in the gene exons. By constructing sgRNA and Cas9 protein expression vectors, which were then transcribed in vitro to obtain sgRNA and Cas9 mRNA, the author microinjected Cas9 mRNA mixed with sgRNA into single cell embryos. Then, using polymerase chain reaction, enzyme digestion and other common techniques to identify sgRNA with mutation efficiency and chimaera F0. Finally, the author crossed F0 with wild type ABSR zebrafish and did further selection to obtain foxp3a frameshifted heterozygotes F1 (foxp3a^+/-) in germline. The acquirement of heterozygotes F1 and foxp3a homozygous mutants F2 (foxp3a^-/-) in future will provide help for further research on T regulatory cells and fish immune system.