TSC1转基因敲除小鼠动物模型的构建及敲除效果的初步研究

Construction of TSC1 transgenic knockout mouse model and their knockout effect

目的为探讨mTOR信号通路在骨骼发育过程中的机制作用,建立骨骼发育相关的TSC1转基因小鼠,提供稳定的动物模型。方法取8周龄健康清洁级TSC1flox/flox小鼠分别与肢芽干细胞特异性重组酶(Prx-1-Cre)小鼠、软骨细胞特异性重组酶(Col2al-Cre)小鼠及成骨细胞特异性重组酶(Osx-Cre)小鼠进行杂交。将繁殖出的小鼠继续与TSC1flox/flox小鼠回交,并对其子代小鼠的基因型进行鉴定及mTOR活性检测。结果杂交后分别获得肢芽干细胞特异性TSC1敲除小鼠、软骨细胞特异性TSC1敲除小鼠和成骨细胞特异性TSC1敲除小鼠各8只。与正常组比较,上述3种转基因小鼠p-S6均比正常组升高(P<0.05)。结论本实验成功应用Cre/loxP系统构建肢芽干细胞特异性TSC1敲除小鼠、软骨细胞特异性TSC1敲除小鼠、成骨细胞特异性TSC1敲除小鼠,均提示mTOR活性增高,有明显TSC1敲除效果,为mTOR信号通路研究骨与软骨发育的机制作用提供实验基础。

Objective The research is aimed to establish TSC1 transgenic mice about bone development,and to provide a stable animal model for investigating the mechanism of mTOR signaling pathway in bone development.Methods8-week-old healthy TSC1 flox/flox mice hybridize with limb bud stem cell specific recombinase(Prx-1-Cre)chondrocyte specific recombinant mice,chondrocyte(Col2 al-Cre)mice and osteoblast specific recombinase(Osx-Cre)mice.The breeding mice are continued to hybridize with TSC1 flox/flox mice,and the genotype and mTOR activity of their offspring mice is identified by PCR.Results After hybridization,we obtain 8 limb bud stem cell specific TSC1 knockout mice,chondrocyte specific TSC1 knockout mice and osteoblast specific TSC1 knockout mice.And p-S6 of those 3 transgenic mice is higher than the normal group(P<0.05).Conclusion Limb bud stem cell specific TSC1 knockout mice,chondrocyte specific TSC1 knockout mice and osteoblast specific TSC1 knockout mice are obtained successfully by Cre/loxP system.They show significantly high mTOR activity,and have a significan TSC1 knockdown effect,providing an experimental basis for study about the mechanism bone and cartilage development by mTOR signaling pathway.