软骨细胞条件性敲除PTEN基因的FGFR3功能增强型点突变小鼠的获得及其表型初步分析  

Initial phenotype analysis on chondrocyte PTEN conditional knockout mice generation from FGFR3 gain of function mutant

作　　者：雷子贤[1] 戚华兵[1] 苏楠[1] 赵子瑜[1] 陈锚锚[1] 何启芬[1] 赵玲[1] 金旻[1] 谢杨丽[1] 李福兵[1] 杜晓兰[1] 陈林[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所全军战创伤中心,创伤、烧伤与复合伤国家重点实验室

出　　处：《中国骨质疏松杂志》2009年第1期9-15,共7页Chinese Journal of Osteoporosis

基　　金：国家重点基础研究发展规划项目(2005CB522604);国家自然基金杰出青年基金资助项目(30425023);国家自然科学基金重点资助项目(30530410)

摘　　要：目的获得在软骨细胞中条件性敲除PTEN基因的FGFR3增强型点突变小鼠并进行初步表型分析。方法通过PTEN条件性基因敲除小鼠（Pten^flox/flox小鼠），与软骨细胞特异表达Cre重组酶的小鼠（Col2αCre）交配，获得在软骨细胞中特异敲除PTEN基因小鼠（Co12αCre：Pten^flox/flox）；同时，利用Pten^flox/flox小鼠与FGFR3增强型点突变小鼠（Fgfr3^G369C/小鼠，即ACH小鼠）交配，子代杂合子小鼠（Pten^flox/＋：ACH）之间再交配，获得Pten^flox/flox：ACH小鼠；通过Col2αCre：Pten^flox/flox和Pten^flox/flox：ACH交配即可获得在软骨细胞中特异性敲除PTEN基因的FGFR3增强型点突变小鼠（Col2αCre：Pten^flox/flox：ACH）。采用PCR对小鼠基因型进行鉴定，免疫荧光染色检测PTEN基因的敲除效率，通过X光平片摄影对小鼠的表型进行初步分析。结果获得了在软骨细胞中敲除PTEN的FGFR3点突变小鼠，免疫荧光染色证实Col2αCre：Pten^flox/flox：ACH小鼠软骨细胞中PTEN蛋白因为基因敲除而表达很低。初步的表型分析显示：Col2αCre：Pten^flox/flox：ACH小鼠身长、尾长均较Pten^flox/flox：ACH小鼠长（P〈0．05，n=5）。Col2αCre：Pten^flox/flox：ACH小鼠表型，较Pten^flox/flox：ACH小鼠的侏儒表型有所缓解。结论采用基于Cre／LoxP系统的条件性基因敲除策略，获得了在软骨细胞中敲除PTEN基因的FGFR3增强型突变小鼠，表型分析初步显示，软骨细胞特异性敲除PTEN基因可部分缓解由FGFR3增强型点突变所导致的小鼠侏儒表型。该小鼠的获得，为研究P13K／AKT信号通路在FGFR3突变介导的软骨生长抑制中的作用提供了实验动物平台。Objecti（Col2αCre： Pten^flox/floxe To obtain the chondrocyte PTEN conditional knockout mice from FGFR3 gain of function mutants and initial phenotype analysis. Methods To produce the chondrocyte PTEN conditional knockout mice, the Pten^flox/flox mice were crossed with Col2αCre mice expressing Cre recombinase in chondrocyte specifically; to produce the Pten^flox/flox： ACH mice, Pten^flox/flox mice were crossed with FGFR3 gain of function mutant mice （Fgfr3^G369C/ mice） which presents a ACH genotype, the daughter heterozygote mice then cross with each other; Finally, the chondrocyte PTEN conditional knockout mice with FGFR3 gain of function mutant （Col2αCre： Pten^flox/flox ：ACH） were obtained by crossing （Col2αCre： Pten^flox/flox and Pten^flox/flox ： ACH. All the daughter mice were identified with PCR assay, PTEN knockout effect was detected with immunofluorescent staining, and phenotypes difference between genotypes were compared with X-ray plate film photography. Results Chondroc（Col2αCre： Pten^flox/floxte PTEN conditional knockout in FGFR3 gain of function mutant mice were obtained. It has been confirmed by immunofluorescent staining that there is few PTEN expression in the ehondrocytes of （Col2αCre： Pten^flox/flox ： ACH mice due to the gene conditional knockout. Preliminary phenotype analysis demonstrates that the mice possessing （Col2αCre： Pten^flox/flox：ACH genotype has longer body and tail length than Pten^flox/flox：ACH mice. Compared with Ptenn^flox/flox：ACH mice, the achondroplasia phenotype of Col2αCre： Pten^flox/flox：ACH mice has been ameliorati（Col2αCre： Pten^flox/floxe. Conclusion Based on Cre/LoxP recombination knockout strategy, （Col2αCre： Pten^flox/flox： ACH mice were obtained. Phenotype analysis re（Col2αCre： Pten^flox/flox ealed that PTEN gene conditional knockout strategy could meliorate the achodroplasia phenotype caused by FGFR3 gain of function mutations. The har（Col2αCre： Pten^flox/floxest of （Col2αCre： Pten^flox

关 键 词：PTEN FGFR3 软骨发育不全 条件性基因敲除 小鼠 

分 类 号：R681.1[医药卫生—骨科学]