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Application of a double mutant zebrafish in the preparation of an animal model of osteopetrosis

A technology of osteosclerosis and zebrafish, which is applied in the application field of double mutant zebrafish in the preparation of animal models of osteosclerosis, can solve the problems of lack of animal models of osteosclerosis, brain malformation, osteosclerosis, etc., and achieves convenient breeding. Easy, high spawn, low cost effect

Active Publication Date: 2021-08-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

More importantly, several recent clinical family studies have found that CSF-1R biallelic mutations can lead to severe brain malformations and osteopetrosis
Although PU.1 and FMS have been reported to play a role in osteoclastopenic osteopetrosis in mice, the mechanisms and interactions that specifically affect osteoclast development remain unknown.
And due to the cost of mouse models, breeding scale and operational limitations, there is still a lack of animal models of osteosclerosis that can be used for osteosclerosis drug evaluation and large-scale candidate compound screening

Method used

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  • Application of a double mutant zebrafish in the preparation of an animal model of osteopetrosis
  • Application of a double mutant zebrafish in the preparation of an animal model of osteopetrosis
  • Application of a double mutant zebrafish in the preparation of an animal model of osteopetrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Pu.1 and FMS have a synergistic effect in the development of refunctional osteoclasts in zebrafish It is known that in mammals, osteoclasts are derived from macrophages. Both PU.1 and FMS are expressed in macrophages, and the abnormality of either gene will lead to abnormal osteoclasts, suggesting that they play an important role in osteoclast formation and maturation. However, whether PU.1 and FMS have a synergistic effect in osteoclast development is unknown. To answer this question, we conducted the following experiments:

[0082] 1) Frozen section experiment: zebrafish specimens were first fixed with 4% PFA (paraformaldehyde) (the fixation time depends on the size of the tissue, such as small fish tissues at room temperature for 2 hours, adult fish tissues at room temperature for 2 days), and then 30% (w / v) sucrose for dehydration (overnight dehydration at 4°C). After the tissue is completely dehydrated and sinks to the bottom of the tube, it can be emb...

Embodiment 2

[0086] Example 2: Compared with FMS, PU.1 mainly acts on zebrafish osteoclastogenesis

[0087] There are two possible reasons for the reduction of functional osteoclasts: one is a disorder of osteoclastogenesis, and the other is a disorder of multinucleated osteoclast maturation. To determine the cause of the reduced functional osteoclasts caused by PU.1 and FMS deficiency, we performed the following experiments:

[0088] The co-staining method of LCP and DAPI immunofluorescence is as follows:

[0089] ①The zebrafish were killed in ice water first, and then the body segment between the pectoral fin and pelvic fin was taken out with a scalpel under a dissecting microscope;

[0090] ② Fix the tissue in 4% PFA overnight;

[0091] ③The removed body was washed with PBST for 3×5mins,

[0092] ④Reuse 20mg / mL cathepsin (name: proteinase K; brand: Fermentas, ThermoFisher Scientific) to digest at room temperature for 2h;

[0093] ⑤ After that, wash with PBST for 3×5mins;

[0094] ⑥...

Embodiment 3

[0102] Example 3: Osteoclast reduction is partially caused by apoptosis

[0103] To identify the cellular mechanism by which pu.1 and fms deficiencies lead to osteoclast reduction, we performed co-staining of LCP with terminal deoxynucleotidyl transferase dUTP-labeled end-label (TUNEL) as follows:

[0104] (1) TUNEL staining:

[0105] ①The zebrafish were killed in ice water first, and then the body segment between the pectoral fin and pelvic fin was taken out with a scalpel under a dissecting microscope;

[0106] ②According to the instructions of the TUNEL kit (name: In-Situ Cell Death Detection Kit TMR red; brand: Roche; address: Basel, Switzerland; article number: 12156792910), wash the removed body with PBST for 3×5mins;

[0107] ③Use 20 mg / mL cathepsin (name: proteinase K; brand: Fermentas, ThermoFisher Scientific) to digest at room temperature for 2 hours;

[0108] ④ Wash with PBST for 3×5mins;

[0109] ⑤Use acetone:ethanol (1:2, v / v) solution to soak for 7 minutes at ...

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Abstract

The invention discloses the application of a double mutant zebrafish in preparing an animal model of osteopetrosis. The double mutant zebrafish is pu.1 G242D ; fms j4e1 Mutant zebrafish, which is a mutant in which the 242nd amino acid glycine in the DAB domain of the zebrafish pu.1 gene is replaced by aspartic acid, and the 614th amino acid valine in the kinase domain of the fms gene is substituted by methionine zebrafish. The double mutant zebrafish of the present invention has symptoms of osteopetrosis similar to humans, so it can be used as an animal model for osteosclerosis drug evaluation and large-scale candidate drug screening. In addition, the double mutant zebrafish of the present invention The scale can be used as a fast reading and processing method for drug screening of osteosclerosis, and has the characteristics of high throughput, short cycle, low cost and convenient operation, and provides an effective way for drug screening of osteosclerosis.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the application of a double mutant zebrafish in preparing an animal model of osteopetrosis. Background technique [0002] Bone development and bone remodeling programs are controlled by a precise balance between the bone formation function of osteoblasts (Ob) (forming new bone) and the bone resorption function of osteoclasts (Oclasts (Oc) (absorbing and digesting old bone). Completed. Bone resorption and bone formation mainly depend on the activities of Oc and Ob. The formation and maturation of Ob and Oc depend largely on transcription factors and cytokines. Therefore, the abnormality of these factors will break the balance between bone formation and bone resorption, resulting in osteoporosis (bone resorption > bone formation) and the occurrence of osteosclerotic diseases (bone formation > bone resorption). [0003] Osteosclerosis (also known as osteopet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00
CPCA01K67/0275A01K2217/03A01K2227/40A01K2267/03A61K49/0008
Inventor 刘伟张译月张文清黄志斌
Owner SOUTH CHINA UNIV OF TECH
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