ABSTRACT
BACKGROUND: Studies have shown that Sonic Hedgehog (Shh) is expressed in mouse Notochordal cells, and India Hedgehog (Ihh) is mainly expressed in chondrocyte-like cells and end plates of mouse embryonic vertebral bodies. However, the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo is unclear. OBJECTIVE: To investigate the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo. METHODS: Male Shh-CreERT2, R26-mTmG/+ mice mated with female R26-mTmG/+ mice to obtain pregnant rats at different periods of pregnancy (E8.5, E11.5, E12.5, E14.5, E16.5, E18.5), followed by injection of 10 g/L tamoxifen at a dose of 10 μL/g. Intervertebral disc tissues were isolated from the mouse embryo at P0, followed by genotype identification. At the same time, male C57BL/6 mice mated with female C57BL/6 mice to obtain pregnant rats at different periods of pregnancy (E11.5, E12.5, E14.5, E16.5, E18.5), and the intervertebral disc tissues of mouse embryo were taken and analyzed. The expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo was detected using immunofluorescence and immunohistochemistry, respectively. Implementation of animal experiments was approved by the Animal Ethics Committee of Soochow University. RESULTS AND CONCLUSION: Shh-CreERT2; R26-mTmG/+ mouse embryos were identified by PCR amplification. The results of immunofluorescent staining showed a gradual decrease in the expression of Shh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. The results of immunohistochemical staining showed a gradual increase in expression of Ihh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. In summary, Shh and Ihh are dynamically expressed during the formation of intervertebral discs in mouse embryo, and provide a basis for further research on the molecular mechanism of intervertebral disc degeneration.
ABSTRACT
In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.