Generation Of Dip2a-LacZ Reporter Mouse Model And Analyzing Dip2a Expression Pattern

Disconnected(disco)-interacting protein 2 homolog A(DIP2A)is evolutionarily conserved protein encoded by Dip2a gene,which belongs to the DIP2 protein family.In vitro based studies suggested that DIP2A is expressed in restricted regions of the nervous system during embryonic development,and may function as a candidate receptor of FSTL1 mediating protective effects on cardio-cerebrovascular.However,the physiological function of endogenous DIP2A is not yet clear.Here our objective is to generate Dip2a-LacZ knockin reporter mouse model by CRISPR/Cas9 system through microinjection and then characterize expression pattern of Dip2a in adult mouse nervous system and its potential function in vivo.The Gene targeting strategy was that pX330-sgRNA plasmid can induce double-stranded breaks,which is just two nucleotides ahead of start codon,the ATG site in exon 1,and then the LacZ-NEO fragment can be integrated into the targeting site with the help of homology arms of Dip2a-LacZ knockin Donor vector.Consequently,LacZ expression can be driven by Dip2a promoter,which at the same time leads to complete inactivation of Dip2a.Then,we detected the correct integration of LacZ-NEO fragment by PCR of the left arm,right arm and LacZ knock in fragment.We also confirmed the inactivation of Dip2a by RT-PCR and Western blot at the RNA and protein expression levels respectively.RT-PCR?LacZ staining and Realtime PCR analysis showed that the LacZ reporter gene expression is identical to the endogenous Dip2a expression.Above all,we successfully generated the LacZ-Dip2a reporter mouse model.Notably,targeted insertion of LacZ reporter gene with NEO cassette was 11.1%(2/18)of live pups and the knockin allele is germline transmitted.Furthermore,knockout of Dip2a doesn't lead to embryonic lethality and Dip2a knockout mice don't show any overt developmental defects.LacZ staining results showed that Dip2a is highly expressed in adult mouse nervous system,including cerebellum(granule cell layer and Purkinje cell layer),hippocampus(dentate granule layer and hippocampal pyramidal layer),spinal cord and retina(outer nuclear layer,inner nuclear layer and ganglion cell).In this study,we made insertion of LacZ reporter gene larger than 5kb by co-microinjection of circular plasmids and eventually we generated Dip2a-LacZ re porter mouse model,which will provide a mouse tool for future study of physiol ogical function of Dip2a in vivo.Also,we hope that our descriptions of Dip2a e xpression pattern in adult mouse nervous system and analysis of its potential rol es will provide a theoretical basis for future research on Dip2a function in the n ervous system.