Generation Of MSpag9 Conditional Knockout Mice And Phenotype Identification

Background:c-Jun NH2-terminal kinase-associated leucine zipper protein（JLP）is a family member of c-Jun NH2-terminal kinase-associated interacting proteins（JIPs）.JLP is a scaffolding protein in JNK and p38 MAP kinase signaling pathway,found by Reddy in 2002.JLP is encoded by SPAG9 gene,which generates three splice variants namely,JLP（3,921 bp;1307 amino acids）,JIP4（3426 bp;1142 amino acids）,and SPAG9（2,268 bp;766 amino acids）.Of these splice variants,JLP is ubiquitously expressed and provide a scaffold function for both JNK and p38MAPK.Several studies have reported the overexpression of SPAG9 gene product in many cancers.However,the function of JLP and in kidney diseases is not clear.Objective:We have found that JLP plays an essential role in renal fibrosis.Here to estabilish the JLP conditional knockout mice to study further mechanism of JLP in kidney diseases.Methords:The conditional knockout vector targeting exon 14 and Exon 18 of mSpag9 were generated by molecular cloning,and then electroporation of embryonic stem（ES）cells was carried out.The targeted ES cells wre screened by PCR and Southern blot,and the correct targeted ES cells were microinjected into blastula of C57BL/6 mice.Chimerical mice were generated after transplanted the blastula into the host mice,and then the mSpag9-Lox P transgenic mice were generated.mSpag9c KO mice could be obtained by crossing between KSP-Cre mice and mSpag9 c KO mice Using western blot and immunohistochemistry to test the expression level of scaffold protein JLP in different organs or tissues.Ovserving and recording the body weight,food intake and urine volume of mice with different genotypes.Results:Ten positive ES cells were cloned by PCR,and 5 were identified by Southern blot analysis.After microinjection we got Chimera mice.Crossed with FLP mice we got 4 male mice and 5 female mice,the genotype of them are all mSpag9^flox/+.Self-crossing the mSpag9^flox/+mice we got 3 mSpag9^flox/floxmice.The genotype of the3 mice were identified by PCR.Using the KSP-Cre mice to generate mSpag9^flox/+and Cre⁺mice.Then backcrossing the mSpag9^flox/floxmice with the mSpag9^flox/+and Cre⁺mice finally we got the mSpag9^flox/flox and Cre⁺mice.The results of western blot and immunohistochemistry show that there is significant difference of JLP among brain,testis,heart,spleen,lung,comparing with kidney.We also found that the difference exist in the kidney from wildtype mice and mSpag9^flox/flox-Cre⁺mice.This indicates that scaffold protein JLP is successfully knockout in kidney and this c KO model is completed.Compared with the other type mice,the growth,development and fertility of kidney specific knockout mice of mSpag9 gene were not affected by gene knockout.There was no significant difference in food intake,body weight,and urine volume in the gene-specific knockout mice.Conclusion:The mSpag9 conditional knockout mice were successfully identified by PCR,western blot and immunohistochemistry which provided the fundation for further study of mSpag9 in vivo.