Generate Dip2 Gene Knockout In Mouse Embryonic Stem Cells

Disco-interacting protein homologue protein(DIP2),can interact with certain neurons in the visual system of Drosophila.Dip2 protein families are highly conserved during evolution,suggesting they may play important biologic roles.From the study,we have found that the mammalian DIP2 protein family is composed of three members:DIP2A,DIP2B and DIP2C.Studies have shown that these three members are inextricably linked,They have high similarities in amino acid sequence and protein structure,They are called interacting proteins.Their structure contains three common regions:DMAP-binding domain,AMP-binding domain and Cai C-binding domain.However,the downstream targets of these domains are not clear and studies on their biologic functions are still missing.Currently,there are very few studies on the DIP2 protein family,with only about 30 published articles.Among them,the research on DIP2A protein is relatively limited.DIP2A protein is a type of protein encoded by Dip2a gene.Previous studies in mice found that Dip2a gene is related to synapse formation,neural development,metabolic immunity and processes in the mouse nervous system.However,the role of DIP2A in the brain and embryonic development is still largely unknown.DIP2B is also a member of the DIP2protein family.At present,most studies on DIP2B use bioinformatics prediction.Based on the results,it is speculated that DIP2B may be associated with human psychiatric diseases,but its endogenous expression mechanism has not been reported.There are few studies on DIP2C protein,and literature studies have shown that Dip2c gene is related to the occurrence of immune metabolism and cardiovascular diseases.In the study of autism spectrum verification,it was discovered that DIP2C is also a susceptibility factor of autism.Its absence can lead to the occurrence of mental disorders,but its specific biological mechanism is still unclear.We generated a Dip2a,Dip2b and Dip2c homozygous knockout 46C ESC cell line using CRISPR/Cas9 genome editing technology under the control of46C^Sox1-gfp embryonic stem cells.Sox1 is the earliest neural precursor-specific marker in mouse embryos,can be used to study the neural differentiation process of stem cells.The eighth exon of Dip2 gene was replaced,insert marker that facilitates subsequent screening.We constructed Dip2a,Dip2b,Dip2c gene knockout mouse embryonic stem cell models,in order to study the influence of Dip2a,Dip2b and Dip2c genes in the early embryonic development process.According to the experimental results,knocking out the Dip2a,Dip2b and Dip2c genes has a significant promotion effect on the differentiation of embryonic stem cells into the endoderm,while it has a strong inhibitory effect on the differentiation of the ectoderm(neurodermal).It shows that knocking out Dip2a,Dip2b and Dip2c genes all have an impact on the neural differ-entiation process of embryonic stem cells.Next,the Dip2 gene family co-expression network can be constructed by transcriptome sequencing.Using the similarity of expression between members to predict new biological functions,it is also possible to find out the regulatory mechanism of different alternative splices of Dip2a,Dip2b and Dip2c genes,and infer more physiological characteristics of the Dip2 gene family.It is also possible to find out the proteins that interact with the DIP2 protein family and key targets by interacting protein profiles,provide drug targets for the treatment of clinically related diseases.