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Zebra fish model for Bernard-Soulier syndrome

A technology of zebrafish and platelets, which is applied to peptide sources, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problem of uneven bleeding and achieve high egg production, low feeding costs, and low cost Effect

Active Publication Date: 2021-09-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deficiency of GPIb-IX-V causes platelets to not adhere to the vascular endothelium normally, leading to skin, mucous membrane bleeding or internal bleeding, with varying degrees of bleeding

Method used

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  • Zebra fish model for Bernard-Soulier syndrome
  • Zebra fish model for Bernard-Soulier syndrome
  • Zebra fish model for Bernard-Soulier syndrome

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Materials and methods:

[0040] (1) Zebrafish farming

[0041] Zebrafish were cultured as described in the literature (Westerfield M: The zebrafish: guide for the laboratory use of zebrafish (Brachdanio rerio). Edition by Eugene, OR, M. Westerfield, 1993).

[0042] (2) The following strains are used in the present invention: AB wild-type zebrafish, gp9 SMU15 Mutant zebrafish, Tg(CD41:GFP) transgenic zebrafish. Among them, each transgenic zebrafish line was combined with gp9 SMU15 Crossbreeding of mutant zebrafish followed by selfing to obtain gp9 in a CD41 transgenic background SMU15 Mutant zebrafish such as Tg(CD41:GFP); gp9- / -.

[0043] (3) CRISPR / Cas9-targeted gene knockout of gp9 gene (refer to figure 1 shown)

[0044] Select the sequence after the ATG start codon of exon 2 of the gp9 gene as the sequence for targeted knockout. According to the http: / / www.crisprscan.org / website, the predicted score is high, the target efficiency is high, and the off-tar...

Embodiment 2

[0047] Detection of gp9 by whole-mount in situ hybridization SMU15 Expression of mutant platelet-related genes

[0048] The following standard experimental procedures were followed (Thisse C, Thisse B.(2008).High-resolution in situ hybridization to whole-mount zebrafish embryos.NATUREPROTOCOLS.VOL.3NO.1).

[0049] The result is as figure 2 part C of figure 2 As shown in part D, in 3dpf, gp9 SMU15 The platelet marker cd41 signal in the mutant is less than that of the wild type, indicating that gp9 SMU15 Mutant platelet counts may be reduced.

Embodiment 3

[0050] Embodiment 3Q-PCR detects gp9 SMU15 Expression of mutant platelet-related genes

[0051] Collect 3dpf WT and 3dpfgp9 SMU15 For the mutant fish, use Tripure RNA Isolation Reagent (Thermo, 15596018) to extract the total RNA of the embryo, and follow the instructions in the instructions. cDNA was reverse transcribed with M-MLV reverse transcriptase (Promega, M1701). Zebrafish elf1a was used as an internal reference gene for Q-PCR detection, and the sequence information of the primers is shown in Table 1 below.

[0052] Table 1

[0053]

[0054]

[0055] The result is as figure 2 As shown in part E, in 3dpf, gp9 SMU15 Compared with the wild type, the expression of platelet-related genes cd41, nfe2, and fog1 in the mutant decreased. Combined with the results of whole-body in situ hybridization of cd41, it further explained that in the embryonic period, gp9 SMU15 The number of platelets is reduced in the mutants.

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Abstract

The invention discloses a zebra fish model for a Bernard-Soulier syndrome. The zebra fish model is a gp9SMU15 mutant zebra fish. According to the zebra fish model, it is found for the first time that gp9 mutation on zebra fish is related to platelet proliferation, the zebra fish gp9SMU15 mutant has similar symptoms of the Bernard-Soulier syndrome to human, and it is proved that the gp9SMU15 mutant zebra fish has blood coagulation dysfunction. In addition, a Bernard-Sourlier Syndrome disease model of thrombocytopenia and blood coagulation dysfunction is established for the first time according to the zebra fish model, and the zebra fish model can become a new carrier for researching the Bernard-Sourlier Syndrome disease and screening drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the field of research on platelet development, in particular to a zebrafish model of giant platelet syndrome. Background technique [0002] Platelets are a major component of the blood system. Platelets play a role in stopping bleeding and clotting when blood vessels are damaged. When the number of platelets decreases or the function decreases, it will lead to the body's hemostatic dysfunction and poor thrombus formation, and cause related platelet diseases. Giant platelet syndrome, also known as Bernard-Soulier Syndrome, is a rare hereditary bleeding disorder with an incidence of less than 1 / 1 million, mainly due to the synthesis of the glycoprotein complex GPIb-IX-V on the platelet membrane and Defective expression or genetic defects of any of the subunits GPIb, GPIX and GPV. Deficiency of GPIb-IX-V causes platelets to fail to adhere to the vascular endothelium normally, leading...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A01K67/027
CPCC12N15/113C12N15/8509C07K14/47A01K67/0276C12N2310/20A01K2207/15A01K2217/075A01K2227/40A01K2267/0306
Inventor 张译月林青周日阳孟盼盼吴亮亮
Owner SOUTH CHINA UNIV OF TECH
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