Fgfr3基因374点突变致软骨发育不全小鼠模型的建立与分析

目的建立模拟人成纤维细胞生长因子3型受体(fibroblastgrowthfactorreceptor3,Fgfr3)374号甘氨酸至精氨酸点突变小鼠模型并进行初步表型分析。方法应用双重PCR法及分子克隆技术构建小鼠Fgfr3-Gly374Arg点突变打靶载体,对胚胎干细胞(embryonicstemcells,ES细胞)进行打靶后,采用G418、Ganciclovir正负双向选择和Southern杂交对打靶ES细胞进行筛选,选发生正确同源重组的ES细胞进行胚泡显微注射,携带Neo基因的Fgfr3-Gly374Arg点突变鼠与EIIa-Cre转基因鼠杂交后,得到只含Fgfr3-Gly374Arg点突变鼠。用PCR法进行了基因型鉴定,并应用骨骼染色、组织学等技术对其表型进行了初步分析。结果Fgfr3-Gly374Arg点突变小鼠体小尾短、头大,前额圆凸,骨骺生长板区明显缩短、结构紊乱,肥大软骨细胞区也有明显减少。同时伴有多数雌性小鼠不孕、子宫、卵巢变小、乳腺发育不良等变化。结论成功地建立了模拟人Fgfr3-Gly374Arg点突变所致软骨发育不全的小鼠模型。

Gly374Arg mutation in Fgfr3 causes achondroplasia in mice

Objective To establish the mouse model of Gly374Arg mutation in fibroblast growth factor receptor 3 ( Fgfr3) and to analyze the phenotype of the mutant mice. Methods The double PCR was used to introduce Gly374Arg point mutation into mouse Fgfr3. The electroporation of embryonic stem(ES) cells was carried out with targeting vector. The targeted ES cells were screened by Positive Negative Selection of G418 and Ganciclovir, and Southern blot. The correct targeted ES cells were microinjected into blastula. Finally, mutant mice were obtained by crossing between EIIa Cre transgeneic mice and mice carrying recombined mutant Fgfr3 allele. The mice were genotyped by PCR, and phenotype was observed by skeleton staining, histology, etc. Results Fgfr3 Gly374Arg mutant mice exhibited small size, short tail, macrocephaly and had dome shaped heads, the epiphyseal growth plates of mutant mice were narrower, and the hypertrophic chondrocyte zone was also obviously decreased. Meanwhile, the majority of female mice were infertile, and the uterus, ovary and mammal gland in mutant female mice were also smaller and underdeveloped. Conclusion The model of Fgfr3 Gly374Arg mutation causing achondroplasia in mice has been established successfully.