Method for evaluating gene variation pathogenicity through mutant cell migration function and application thereof

A technology of gene mutation and cell migration, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as the incomplete realization of the advantages of the zebrafish model, and achieve large-scale screening, convenient data collection and preservation, and high accuracy. with specific effects

Active Publication Date: 2021-10-22
GUANGZHOU KINGMED DIAGNOSTICS CENT +1
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  • Abstract
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Problems solved by technology

Most of the research is carried out based on easy-to-detect indicators such as hemorrhage, cardiac structural abnormalities, and electrophysiology, and these methods can also be carried out in mice. The advantages of zebrafish as a model have not been fully reflected

Method used

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  • Method for evaluating gene variation pathogenicity through mutant cell migration function and application thereof
  • Method for evaluating gene variation pathogenicity through mutant cell migration function and application thereof
  • Method for evaluating gene variation pathogenicity through mutant cell migration function and application thereof

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Experimental program
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Embodiment

[0046] 1. Breeding of zebrafish

[0047] The zebrafish were cultivated according to the method reported in the literature (Westerfield M: The zebrafish: guide for the laboratory use of zebrafish (Brachdanio rerio). Edition by Eugene, OR, M. Westerfield, 1993).

[0048] The following zebrafish strains are used in the present invention:

[0049] ABSR wild-type zebrafish, transgenic line Tg(lyz:DsRed), elmo1 gene deletion zebrafish mutant Tg(lyz:DsRed): from Zilong Wen’s laboratory (L.Li, B.Yan, Y.Q.Shi, W.Q.Zhang, Z.L.Wen, Liveimaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem 287, 25353-25360 (2012)).

[0050] The elmo1 gene-deficient zebrafish mutant was designed using TALEN gene editing technology to design the target site on exon 18 of the elmo1 gene (NC_007130.7). This technique was used to knock out 13 pairs of base pairs (GCCCTCAGGGAGA, SEQ ID NO.1) on the PH domain of the zebrafish Elmo1 protein and Dock2 ...

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Abstract

The invention relates to a method for evaluating gene variation pathogenicity through a mutant cell migration function and an application thereof, and belongs to the technical field of animal model construction. The method comprises the following steps: constructing an expression system of a neutrophil specific expression human ELMO1 gene variation site; cloning a plasmid carrying a to-be-evaluated gene variation site sequence into an ELMO1 gene deletion zebrafish mutant model, and specifically expressing the to-be-evaluated ELMO1 gene variation site in the neutrophil; and carrying out delayed in-vivo imaging: carrying out visual tracking on the neutrophil carrying the ELMO1 gene variation site in the zebra fish gene deletion mutant model through delayed in-vivo imaging, and evaluating the influence of the gene variation site on the migration function of the neutrophil so as to evaluate the pathogenicity of the gene variation site. The method can be used for identifying the functional change of the gene variation based on the animal model, does not depend on a sample of a patient, and has the advantages of convenience in data acquisition and storage and high experiment repeatability and accuracy.

Description

technical field [0001] The invention relates to the technical field of animal model construction, in particular to a method and application for evaluating the pathogenicity of gene variation by using mutant cell migration function. Background technique [0002] The ELMO1 protein translated from the ELMO1 gene interacts with the DOCK2 protein complex and participates in the regulation of cell motility by regulating the activity of the RAC protein. [0003] In the past decade, with the advent of high-throughput genome sequencing, human genome information has rapidly accumulated. The deletion of Dock2 gene has been reported to be associated with congenital immunodeficiency diseases. On the contrary, the analysis of ELMO1 gene polymorphism in different populations around the world found that ELMO1 gene is associated with diabetes, inflammatory arthritis, kidney disease, etc. There is a correlation with autoimmune diseases. Although the ELMO1 gene regulates cell motility throug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/62C12N15/65A01K67/027
CPCC12N15/8509C07K14/47C12N15/65A01K67/0278C12N2800/106C12N2830/008C07K2319/60A01K2207/05A01K2207/15A01K2227/40A01K2267/0393A01K2267/0325A01K2267/0368A01K2267/0362Y02A40/81
Inventor 莫桂玲薛容涛徐进孙明明范喜杰王莹吕美
Owner GUANGZHOU KINGMED DIAGNOSTICS CENT
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