The Establishment of IKBKB Gene c.1183T>C Point Mutation Mice and Analysis of Basic Phenotype

FENG Minxuan^1,2, ZHOU Lina^1,2, QIN Tao^2,3, ZHAO Xiaodong^1,2, JIA Yanjun^1,2*

(¹Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, Chongqing Key Laboratory of Child Infection and Immunity, Chongqing 400014, China; ²Ministry of Education Key Laboratory of Child Development and Disorders; National Clinical Research Center for Child Health and Disorder (Chongqing); China International Science and Technology Cooperation Base of Child development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China; ³Department of Infection, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China)

Abstract:

To explore the effect of IKBKB gene c.1183T>C mutation on immune response, CRISPR/Cas9 gene-targeting strategy was used to introduce a corresponding mutation in the IKBKB gene in C57BL/6J mouse embryos. Genomic DNA was genotyped by PCR and confirmed by Sanger sequencing. Both splenic and thymiclymphocytes were isolated by density centrifugation, and then the mRNA and protein expressions of members of IKKs family were detected by Real-time PCR and Western blot, respectively. The MOE (molecular operating environment) software was used to analyze the PDB structure of the protein and to establish the 3D model. Sanger sequencing results showed that the mutant mouse was accurately generated and normally propagated. In addition, the mRNA expression of IKBKB in splenic and thymic lymphocytes from mutant mice was comparable to that in wild type cells, whereas the expression of IKKβ protein in abovementioned lymphocytes of the IKBKB Y397H mutant mice was obviously decreased. Furthermore, protein structure analysis showed that the conformational structure of mutant IKKβ protein obviously changed. These results showed that the abundance of IKKβ protein was reduced in mutant mice, which might be caused by the disruption and instability of spatial structure of mutant protein. This research provided a good model basis for further exploration of the site mutation to the steady state regulation in immune cells and its pathogenic mechanisms.

CSTR: 32200.14.cjcb.2020.03.0011