Construction Of TOX2 Knockout Mouse Model By CRISPR/Cas9 Technology And Preliminary Study On TOX2 Knockout Mice

Objective: TOX2 is a transcription factor that belongs to the TOX family.Although TOX2 has been shown to play a crucial role in the development of NK cells,little is known about the role of TOX2 in the development of lymphocytes.The use of CRISPR/Cas9 gene editing technique to construct TOX2 knockout mouse model can further study its functions and effects.Methods: CRISPR/Cas9 gene editing technology has become the most rapidly developed gene editing technology due to its advantages of simple design and high efficiency.In this study,TOX2 knockout mice model were successfully constructed by CRISPR/Cas9 gene editing technique.First,according to the relevant principles of CRISPR/Cas9 gene editing technology,single guide RNA was designed.Then,sg RNA was connected to the PX459 plasmid vector.The effectiveness of sg RNA was verified in vitro and intracellular.The proven recombinant plasmid was injected into the fertilized mouse egg.After the homozygous mice were obtained,the homozygous mice could be preliminarily analyzed.Results: In this study,we designed three sg RNAs.The sg RNA1 and sg RNA2 have been shown to be effective in vitro and intracellular.TOX2 knockout mice were obtained by prokaryotic microinjection,and their inheritance was stable.TOX2 knockout mice were successfully constructed.Flow cytometry was used to analyze the proportion of lymphocytes and their subsets in TOX2 knockout mice,and The results showed no significant change.Conclusion: TOX2 gene has no significant effect on T and B lymphocyte development in mice.The successful establishment of TOX2 knockout mouse model provides a important basis for further study on the function of TOX2 gene.