The Construction Of A Gdf6 Mutation Mouse Model And Study On Its Pathogenicity

Objective:Multiple synostoses syndrome 4(SYNS4)is a new type of genetic bone disease which was designated by us.We identified a mutation of GDF6 gene as the etiology in the family.The purpose of this study is to construct a Gdf6-Y443 N mouse model by using CRISPR/Cas9 gene editing technology to study the role of GDF6 gene in the development and maintenance of bones and joints.Methods:(1)Use the CRISPR/Cas9 gene editing technology to construct a Gdf6-Y443 N mouse model on the mouse of C57BL/6J;(2)Study the body coordination ability of the Gdf6-Y443 N wild type,heterozygous and homozygous mice by the experiments of spontaneous activity,rotarod test,pole test,beam balance walking and adjustable variable-speed belt threadmill;(3)Detect the auditory function of the mouse model by auditory brainstem response;(4)Research the bone of the mouse model by alizarin red-alcian blue double staining and micro computed tomography(Micro CT).Results:(1)A Gdf6-Y443 N mouse model was successfully constructed using the CRISPR/Cas9 gene editing technology and obtained a sufficient number of mice by breeding;(2)The homozygous mice performed poorly on rotarod test,pole test,beam balance walking and adjustable variable-speed belt threadmill.(3)The hearing thresholds of the homozygous mice at the sound frequency of 4 kHz and 8 kHz were higher than that of the wild type and heterozygous,suggesting a hearing impairment.There was no significant difference between heterozygous mice and wild type mice;(4)There was no significant difference in the overall appearance of bones between the newborn wild type,heterozygous and homozygous mice,but the carpal and tarsal joints of the homozygous mice fused;(5)The Gdf6-Y443 N mouse model reproduced the phenotype of human multiple synostoses syndrome 4.Conclusion: The Gdf6-Y443 N mouse model was successfully constructed,and the pathogenicity of Gdf6-Y443 N was studied on this animal model.It proved the role of GDF6 in the development and maintenance of bones and joints,and laid a solid foundation for further studying the molecular mechanism of GDF6.