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Construction and application of mouse model with conditional knockout of MACF1 gene in osteoblast
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An osteoblast, mouse model technology, applied in the field of biomedicine, can solve problems such as incomplete response
Inactive Publication Date: 2017-09-01
NORTHWESTERN POLYTECHNICAL UNIV
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However, experiments at the cellular level cannot fully reflect the real situation in the body, and in vivo experiments are needed to verify
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Embodiment 1
[0050] Example 1: Construction of a mouse model in which the MACF1 gene is conditionally knocked out in osteoblasts.
[0051] 1.1 Experimental animals.
[0052] MACF1 fl / + Breeding of mice and Sp7-Cre transgenic mice.
[0053] MACF1 fl / + Mice and Sp7-Cre transgenic mice entered the breeding area after isolation and observation, and no abnormalities were found. The feeding was carried out in accordance with the SPF animal feeding standards, and the experimental operations were strictly carried out in accordance with the relevant regulations of SPF animal management.
[0055] Clip mouse toes and extract MACF1 fl / + Mouse genomic DNA, using primers for mouse genotype identification; using the mouse MACF1 gene sequence as a template, the specific primer sequences are designed as follows:
[0056] AP179: 5'-AAAGAAACGGAAATACTGGCC-3';
[0057] AP180: 5'-GCAGCTTAATTCTGCCAAATTC-3'.
[0058] refer to figure 2 , PCR identification results...
Embodiment 2
[0069] Example 2: Detection of the expression of MACF1 in osteoblasts of a conditional knockout mouse model.
[0070] Newborn mouse calvarial osteoblasts were taken, and their RNA was extracted for SYBR green Real-time Quantitative PCR (qRT-PCR) to detect the expression of MACF1 at the mRNA level, so as to verify the conditional knock-out of MACF1 in osteoblasts. The low expression in osteoblasts of the mouse model was excluded, thus confirming that the animal model was successfully constructed.
[0071] refer to Figure 5 , MACF1 in Sp7-Cre; MACF1 fl / fl The expression level in mouse osteoblasts was significantly lower than that of MACF1 fl / fl mice, indicating that the mouse model for conditional knockout of the MACF1 gene in osteoblasts was successfully constructed.
Embodiment 3
[0072] Example 3: The effect of knocking out MACF1 in osteoblasts on the expression of related osteogenic genes in mouse osteoblasts.
[0073] Newborn mouse calvarial osteoblasts were collected, and their RNA was extracted for qRT-PCR to detect the expression of osteogenic differentiation marker genes (ALP, RUNX2, COL-I, OCN).
[0074] refer to Image 6 , it was shown that, with MACF1 fl / fl Mice compared to Sp7-Cre;MACF1 fl / fl The expression levels of related osteogenic genes in mouse osteoblasts were significantly reduced, indicating that knocking out MACF1 in osteoblasts reduced the differentiation ability of osteoblasts.
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Abstract
The invention discloses a construction and an application of a mouse model with conditional knockout of an MACF1 gene in osteoblast and is used for solving the technical problem that the practicality is poor in the prior art. The technical scheme is that the mouse model (Sp7-Cre; MACF1f1 / f1) with conditional knockout of the MACF1 gene in the osteoblast is obtained by means of a Cre-Loxp system by mating a MACF1f1 / f1 mouse with a Sp7-Cre transgenic mouse, osteoblast of which specifically expressed Cre recombinase. The bone forming ability of the mouse with conditional knockout of the MACF1 gene in the osteoblast is reduced. The model can be used for researching the function of the MACF1 gene in the osteoblast and the effect thereof in a bone forming process, and meanwhile, can be used as an animal model for researching pathogenesis / generation mechanism of osteoporosis or other bone formation related diseases, and is used for screening drugs for preventing, treating or improving the diseases.
Description
technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the construction and application of a mouse model in which MACF1 gene is conditionally knocked out in osteoblasts. Background technique [0002] Osteoblasts (OB) are the main functional cells for bone formation and repair. Osteoblasts can synthesize and secrete collagen and glycoprotein to form bone matrix, and promote the mineralization of matrix to form bone tissue; at the same time, osteoblasts also participate in the regulation of osteoclastic bone resorption and maintain the metabolic balance of bone. Therefore, the functional status of osteoblasts is closely related to bone growth, shaping and reconstruction. Any factor that inhibits the formation and differentiation of osteoblasts and promotes their apoptosis can reduce bone formation, unbalance bone remodeling, and lead to osteopenia. Osteogenesis of osteoblasts is a complex process regulated by multiple genes, an...
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