基于Cre/LoxP系统建立软骨组织特异性印第安刺猬蛋白基因敲除小鼠模型

Establishment of Indian hedgehog protein conditional knockout mouse models based on Cre/LoxP system

背景:印第安刺猬蛋白(Ihh)条件性基因敲除小鼠动物模型的建立,实现了印第安刺猬蛋白条件性基因敲除能够在时间和组织上具有选择性,能够为进一步研究与生长板有关的严重遗传病提供实验基础。目的:基于Cre/LoxP系统建立软骨组织特异性印第安刺猬蛋白基因敲除小鼠模型,并进一步观察其表型变化差异。方法:SPF级亲代Ihh^(fl/fl)小鼠与Col2a1-CreERT2小鼠,均由美国布朗大学魏垒教授赠予。将亲代Ihh^(fl/fl)小鼠与Col2a1-CreERT2小鼠杂交,获得基因型Col2a1-CreERT2,Ihh^(fl/-)小鼠;再将此基因型小鼠自交,获得刚出生的小鼠。通过琼脂糖凝胶电泳对刚出生小鼠进行基因鉴定,随机分为2组:实验组小鼠于出生后第1天开始腹腔注射玉米油溶解的20 g/L他莫昔芬10μL,连续注射3 d;对照组小鼠腹腔注射等量玉米油。出生后第6天RT-qPCR检测印第安刺猬蛋白基因的敲除率;第8天小鼠膝关节行番红固绿染色、Von Kassa染色进行初步表型观察;第8周行小鼠大体形态观察、记录后肢股骨/胫骨长度,后肢X射线片观察。结果与结论:①通过RT-qPCR证实了印第安刺猬蛋白条件性基因敲除率为76.83%,8周龄小鼠大体形态表现为肢体短小畸形(P <0.01);②X射线显示敲除小鼠胫骨明显缩短且骨骺端发育异常;③番红固绿染色显示印第安刺猬蛋白条件性敲除表达在发育中的小鼠胫骨生长板软骨组织这一特定区域;④Von Kassa染色显示Ihh^(d/d)小鼠生长板过早闭合;⑤初步建立了印第安刺猬蛋白条件性基因敲除小鼠模型,印第安刺猬蛋白条件性基因敲除后引起小鼠生长板区软骨细胞增殖紊乱,生长板区软骨细胞提前矿化,生长板过早骨化闭合,小鼠表现为肢体短小畸形。

BACKGROUND: The establishment of an animal model of Indian hedgehog protein(Ihh) conditional knockout mice can realize the time and tissue selectivity of Ihh conditional gene knockout, which can provide an experimental basis for further research on some serious genetic diseases related to growth plates. OBJECTIVE: To establish a cartilage tissue-specific Ihh knockout mouse model by Cre/LoxP system, and further observe the difference in phenotypic changes. METHODS: Ihhfl/fl and Col2a1-CreERT2 mice, SPF grade, were provided by Prof. Wei Lei from Brown University. Ihhfl/fl and Col2a1-CreERT2 mice were hybrided to obtain Col2a1-CreERT2, and Ihhfl/-mice, and then self-matched to obtain newly born mice. Genetic identification of newly born mice was performed by agarose gel electrophoresis, and then randomized into two groups. The mice received intraperitoneal injection of 20 g/L tamoxifen(10 μL) dissolved in corn oil at the first day after born for 3 consecutive days(experimental group). The controls were given the same volume of corn oil. The knockdown rate of the Ihh gene was detected by RT-qPCR at 6 days after birth. The initial phenotype of the mouse knee was observed by Safranin O and Von Kassa staining on day 8. The gross morphology of the 8-week-old mice was observed, the length of femur and tibia in the hind limb was recorded, and the lower extremity was observed by X-ray film. RESULTS AND CONCLUSION:(1) The Ihh conditional gene knockout rate was 76.83% by RT-qPCR. The result of gross morphology showed short limb deformity(P < 0.01).(2) X-ray showed that the tibia of the knockout mice was significantly shortened.(3) Safranin O staining showed that Ihh conditional knockout was expressed in a specific region of the developing mouse tibia growth plate cartilage tissue.(4) Von Kassa staining showed that Ihhd/d mouse growth plates closed prematurely. The Ihh conditional knockout mouse model was initially established.(5) After the Ihh conditional gene knockout, the chondrocyte proliferation disorder in the growth plate area of mice was induced. The cells in the growth plate area were mineralized in advance, the growth plate was prematurely ossified, and the mice showed short limb deformities.