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

doi:10.3969/j.issn.2095-4344.1248

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: tissue construction, bone tissue engineering, cartilage growth plate, conditional gene knockout, animal model, tamoxifen, mice, Ihh

CLC Number:

R446
R318

Yuan Tao1, Wang Yongzhuo1, Huang Yuanzhang1, Xi Gang1, Wei Lei1, 2, Zhang Min1. Establishment of Indian hedgehog protein conditional knockout mouse models based on Cre/LoxP system[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(19): 3013-3018.

Figures/Tables 2

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