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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。
Objective To establish a murine model of point mutation of glycine to arginine at position 374 of fibroblast growth factor receptor 3 (Fgfr3) and perform preliminary phenotypic analysis. Methods Mouse Fgfr3-Gly374Arg point mutation target vector was constructed by double-PCR and molecular cloning techniques. After embryonic stem cells (ES cells) were targeted, G418, Ganciclovir positive and negative bi-directional selection and Southern blotting were used to screen target ES cells , Blastocyst microinjection was performed on ES cells with the correct homologous recombination. Fgfr3-Gly374Arg point-mutated mice carrying Neo gene were crossed with EIIa-Cre transgenic mice to obtain Fgfr3-Gly374Arg point-mutant mice. The genotypes were identified by PCR and their phenotypes were analyzed by skeletal staining and histology. Results Fgfr3-Gly374Arg point mutation mice body short tail, head large, convex forehead, epiphyseal growth plate area was significantly shortened, structural disorders, hypertrophic chondrocytes area also significantly reduced. At the same time with the majority of female mice infertility, uterus, ovarian smaller, dysplasia and other changes in breast. Conclusion The mouse model of hypochondroplasia induced by Fgfr3-Gly374Arg point mutation was established successfully.