Construction Of FosB Knockout Mouse Model By CRISPR/Cas9 Technology And Preliminary Study On Plcg2 (Deletion Of Exon 20-22) Mice

Three families with cold-induced urticaria are indentified carrying a deletion of the exon 20 to 22 of the Phospholipase C?-2?Plcg2?gene deletion on chromosome 16,and this deletion triggers multiple leukocyte subpopulations.In order to further explore the specific mechanism of potential deficiencies in different subsets of lymphocytes caused by mutation of Plcg2,we generated a mouse model with the specific deletion of exon20-22 in Plcg2 with the genetic editing tool CRISPR/Cas9.More thorough and profound studies on Plcg2 exon20-22 deletion mice are planned to reveal the specific role of the domain coded by exon20-22 of Plcg2.Some studies have found that the FosB gene is highly expressed in the germinal center B cells compared with the naive B cells by RNA sequencing.In order to further explore the specific function of the gene in the development of naive B cells and the function of B cells upon antigen challenge,we designed to construct a FosB gene knockout using CRISPR/Cas9.The mouse model thus provides a reliable experimental object for further exploration of related functions and mechanisms.The new generation of gene editing technology CRISPR/Cas9 is a simple and fast gene editing tool,and its application greatly facilitates biological science research.In this study,sgRNA targeting mouse FosB together with Cas9 protein was microinjected into embryos at single cell stage.Deletion of FosB will be confirmed by sequencing of genomic DNA from individual pups generated after microinjection.Next flow cytometry was used to detect and analyze the changes of T,B lymphocytes and subpopulations in each immune organ,and the changes of calcium ion flux was evaluated in B lymphocytes in spleen of Plcg2?exon20-22?mice.The results showed that some of the embryo-injected mouse FosB gene had a frame shift mutation and could be stably inherited in off springs..Compared with Plcg2^+/+,Plcg2^{?20-22/?20-22}had significant changes in the development of B lymphocytes and their subpopulations.There was no sign significant difference in T lymphocytes.The trend of calcium flux was basically the same,but the peak value decreased significantly.However,there was no significant change in the trends of T,B lymphocytes and calcium flux between Plcg2^+/?20-22and Plcg2^+/+.In summary,the FosB knockout mouse model was successfully constructed,and the domain coded by exon 20-22 of Plcg2 has a critical role in the development of B lymphocytes.