CN113528570B - Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application - Google Patents

Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application Download PDF

Info

Publication number
CN113528570B
CN113528570B CN202110779589.XA CN202110779589A CN113528570B CN 113528570 B CN113528570 B CN 113528570B CN 202110779589 A CN202110779589 A CN 202110779589A CN 113528570 B CN113528570 B CN 113528570B
Authority
CN
China
Prior art keywords
elmo1
gene
zebra fish
neutrophils
mutant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110779589.XA
Other languages
Chinese (zh)
Other versions
CN113528570A (en
Inventor
莫桂玲
薛容涛
徐进
孙明明
范喜杰
王莹
吕美
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Guangzhou Kingmed Diagnostics Central Co Ltd
Original Assignee
South China University of Technology SCUT
Guangzhou Kingmed Diagnostics Central Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT, Guangzhou Kingmed Diagnostics Central Co Ltd filed Critical South China University of Technology SCUT
Priority to CN202110779589.XA priority Critical patent/CN113528570B/en
Publication of CN113528570A publication Critical patent/CN113528570A/en
Application granted granted Critical
Publication of CN113528570B publication Critical patent/CN113528570B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0368—Animal model for inflammation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Abstract

The invention relates to a method for evaluating pathogenicity of genetic variation by using a mutant cell migration function and application thereof, belonging to the technical field of animal model construction. The method comprises the following steps: constructing an expression system for specifically expressing human ELMO1 gene mutation sites by neutrophils: cloning a plasmid carrying a gene variation site sequence to be evaluated into an ELMO1 gene deletion zebra fish mutant model, and specifically expressing the gene variation site of the ELMO1 to be evaluated in neutrophils; time-lapse in vivo imaging: and (3) carrying out visual tracking on the neutrophils carrying the ELMO1 gene mutation site in the zebra fish gene deletion mutant model through time-lapse live imaging, and evaluating the influence of the gene mutation site on the migration function of the neutrophils, thereby evaluating the pathogenicity of the gene mutation site. The method can identify the functional variation of the genetic variation based on an animal model, is independent of a patient sample, and has the advantages of convenience in data acquisition and storage and high experimental repeatability and accuracy.

Description

Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application
Technical Field
The invention relates to the technical field of animal model construction, in particular to a method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application thereof.
Background
ELMO1 protein translated from ELMO1 gene interacts with DOCK2 protein complex, and participates in regulating cell movement function by regulating RAC protein activity.
Over the past decade, human genomic information has rapidly accumulated with the advent of high throughput genomic sequencing. On the contrary, the deletion of the Dock2 gene has been reported to be related to congenital immunodeficiency diseases, and analysis of the gene polymorphism of ELMO1 is carried out in different people around the world, so that the ELMO1 gene is related to autoimmune diseases such as diabetes, inflammatory arthritis, kidney diseases and the like. Although ELMO1 gene plays a role in regulating cell movement through ELMO1-DOCK2-RAC1 ternary complex, studies of mice and cell lines show that ELMO1 gene mutation leads to impaired cell migration function, however, in ELMO1 gene-deleted mice, the accumulation amount of neutrophils at chronic inflammation sites is obviously reduced compared with that of wild type. This chemotactic defect of neutrophils, in turn, results in a good prognosis of autoimmune disease. In the study of inflammatory enteritis of Salmonella infection, the ELMO1 gene-deficient mice have reduced bacterial internalization of macrophages, which also results in reduced bacterial burden in the intestinal tract of the mice and the occurrence of inflammatory reactions in the intestinal tract, thus slowing the progression of the disease.
The research on ELMO1 genes in the mouse model combines the analysis of gene polymorphism of the population carrying ELMO1 gene mutation to find that the ELMO1 protein with high expression level can be used as an index of disease progress, and the poor prognosis of the disease is predicted. Bioinformatic data from clinical samples indicate that ELMO1 genes affect disease progression by affecting chemotaxis of immune cells for inflammation.
However, how these ELMO1 gene mutations are present in the human population affect immune cell function, there is still a need for more specific and definitive functional verification to confirm their relationship to disease susceptibility. In previous practice, researchers isolated neutrophils directly from patient whole blood cells and tested these neutrophils carrying ELMO1 mutations for their ability to migrate in vitro to identify their pathogenicity. In addition to in vitro experiments, in vivo functional verification of gene mutants by using animal models is an indispensable strategy for providing more reliable disease consultation for clinic.
In summary, the detection and identification of how to influence the cell movement for the mutation site of the specific gene are currently based on the collection of the original sample of the patient, which is unfavorable for the large-scale screening of the mutation site, and has the advantages of higher cost, low repeatability and larger required manpower and material resources. Therefore, a convenient and accurate animal model is provided for diagnosing and identifying specific genetic variation sites, and the need is felt. Zebra fish, a vertebrate highly conserved with mice and humans, has been successfully constructed into a number of human disease models for large-scale drug screening. However, the research on the pathogenicity of specific genetic variation by using a zebra fish model is still very limited. Most studies were conducted on the basis of easily detected indicators of bleeding, heart structural abnormalities, electrophysiology, etc., which can also be performed in mice, the advantages of zebra fish as a model are not fully achieved.
Disclosure of Invention
Based on the above, it is necessary to provide a method for evaluating pathogenicity of genetic variation by mutant cell migration function, by which the functional variation of genetic variation site can be identified based on animal model, and the method is independent of patient sample, and has the advantages of convenient data collection and preservation, and high experimental repeatability and accuracy.
A method for evaluating pathogenicity of genetic variation with mutant cell migration function, comprising the steps of:
constructing an expression system for specifically expressing human ELMO1 gene mutation sites by neutrophils: cloning plasmids carrying a gene variation site sequence to be evaluated into an ELMO1 gene deletion zebra fish mutant model by a microinjection method, and specifically expressing the ELMO1 gene variation site to be evaluated in neutrophils under the drive of a neutrophil specific promoter lyz so as to obtain a zebra fish gene variation site expression system;
time-lapse in vivo imaging: and (3) carrying out visual tracking on the neutrophils carrying the ELMO1 gene mutation sites in the zebra fish gene deletion mutant model through time-lapse live imaging, and evaluating the influence of the gene mutation sites on the migration function of the neutrophils through the movement record of the neutrophils, thereby evaluating the pathogenicity of the gene mutation sites.
In one embodiment, the migration function of neutrophils in the ELMO1 gene-deleted zebra fish mutant model is determined by expression of human ELMO1 gene mutation sites.
In one embodiment, the ELMO1 gene-deleted zebra fish mutant model is a mutant with a sequence shown as SEQ ID NO.1 knocked out.
In one embodiment, the genetic variation site to be evaluated is: p.E90K, p.D194G or p.R354X.
In one embodiment, in the step of constructing the expression system for specifically expressing the human ELMO1 gene mutation site by using the neutrophil, after the neutrophil specific promoter lyz, the fusion protein of the human ELMO1 protein and the fluorescent protein GFP is expressed, so that the visual tracking of the neutrophil in the expression system of the human ELMO1 gene mutation site is realized.
In one embodiment, in the step of time-lapse live imaging, the migration path and migration rate of neutrophils expressing the variant site of the human ELMO1 gene on the zebra fish yolk sac are recorded.
The invention also discloses application of the method for evaluating the pathogenicity of the genetic variation by using the migration function of the mutant cells in researching pathogenicity of human ELMO1 genetic variation sites.
In one embodiment, the pathogenicity is a result of impaired or increased neutrophil motor function, thereby increasing the susceptibility to an infectious disease or autoimmune disease.
In one embodiment, the increased infectious or autoimmune disease comprises chronic infection, inflammatory bowel disease, rheumatoid arthritis, or diabetes.
The invention also discloses a biomarker for diseases caused by the impaired migration function of neutrophils, wherein the biomarker is p.R354X.
The general conception of the invention is as follows:
1) Constructing a zebra fish ELMO1 gene deletion mutant which can be used for evaluating pathogenicity of human ELMO1 gene mutation sites by using a TALEN gene editing technology, and knocking out 13 pairs of base pairs on a PH structural domain combined by ELMO1 protein and Dock2 protein. The deletion of base pairs causes frame shift mutation, which leads to premature stop codons, leads to the loss of PH structural domains and finally generates gene deletion mutants of the zebra fish Elmo1 protein, which are nonfunctional.
2) After obtaining the zebra fish elmo1 gene deletion mutant, the random movement of the neutrophils on the zebra fish young fish yolk sac three days after birth (3 dpf) was recorded by using a time-lapse in vivo imaging technique in combination with a transgenic line Tg (lyz: dsRed) which is stably expressed in the mutant and can specifically mark the neutrophils. After shooting, tracking and measuring cell tracks by using image analysis processing software imageJ, and statistically comparing the difference between the zebra fish elmo1 gene deletion mutant and the sibling heterozygote, wild zebra fish neutrophil migration functions.
3) Construction of Tg (lyz:elmo 1) ze -P2A-GFP) transient expression system. The P2A self-cleavage peptide is utilized to connect the elmo1 coding region of the zebra fish with fluorescent protein GFP, and the obtained plasmid is injected into single-cell embryo of the zebra fish by microinjection, so that the visualization of the movement process of the neutrophil expressing the elmo1 gene in the juvenile fish of the zebra fish can be realized. After the expression system was established, we recorded random movements of neutrophils on the yolk sac of young zebra fish three days after birth (3 dpf) using a time-lapse in vivo imaging technique. After shooting is completed, tracking and measuring cell tracks by using image analysis processing software imageJ, and statistically analyzing whether the ELMO1 gene regulates and controls neutrophil migration function due to a cell autonomy mechanism, so as to be used as an experimental basis for constructing a human ELMO1 gene mutation site transient expression system to evaluate pathogenicity of the gene mutation site.
4) Construction of Tg(lyz:ELMO1 hu-WT GFP) transient expression system. The coding region of the human ELMO1 gene and fluorescent protein GFP are directly connected to form ELMO1-GFP fusion protein, and the obtained plasmid is injected into the single-cell embryo of the zebra fish by a microinjection method, so that the visual tracking of the neutrophil expressing the human ELMO1 gene is realized. After the expression system was established, we recorded random movements of neutrophils on the yolk sac of young zebra fish three days after birth (3 dpf) using a time-lapse in vivo imaging technique. After shooting is completed, tracking and measuring cell tracks by using image analysis processing software imageJ, and statistically analyzing whether the human ELMO1 gene is highly conserved with the zebra fish ELMO1 gene, so that the function of regulating and controlling neutrophil migration can be realized, and the method is used as an experimental basis for constructing a human ELMO1 gene mutation site transient expression system to evaluate pathogenicity of a gene mutation site.
5) Construction of Tg (lyz: ELMO 1) hu-VUS GFP) transient expression system. We have three human ELMO1 gene mutation sites involved in the present invention: p.E90K (c.268G)>A)、p.D194G(c.581A>G) And p.R354X (c.1060C)>T) coding region and fluorescent protein GFP are directly connected to form ELMO1 hu-VUS GFP fusion protein, and injecting the obtained plasmid into the single cell embryo of zebra fish by microinjection method, thereby realizing the visual tracking of the neutrophil expressing the human ELMO1 gene mutation site. After the expression system was established, we recorded random movements of neutrophils on the yolk sac of young zebra fish three days after birth (3 dpf) using a time-lapse in vivo imaging technique. After shooting, the cell trails are tracked and measured by using image analysis processing software ImageJ, and whether the expression of the genetic variation sites causes the change of the migration function of neutrophils is statistically analyzed, so that the pathogenicity of the genetic variation sites is evaluated.
Compared with the prior art, the invention has the following beneficial effects:
the method for evaluating the pathogenicity of the genetic variation by using the cell migration function of the mutant utilizes the zebra fish as an animal model to perform functional verification of the genetic variation site, has the characteristics of low cost and short experimental period, and is more convenient for large-scale screening compared with a mouse model.
According to the invention, through the over-expression experiments of the specific gene mutation sites of the zebra fish and the wild zebra fish, the change of the cell functions after the mutation is observed in vivo, and the pathogenicity of the gene mutation sites is more accurately evaluated, so that the method shows that the movement speed of the neutrophils of the zebra elmo1 gene mutant is obviously reduced, and the method can be used for evaluating the influence of the gene mutation on the movement speed of the granulocytes
The method can be used for over-expression of various variants of specific genes in specific cells (neutrophils), can be used for evaluating the functional change of the gene mutation on a specific cell, and has higher accuracy and specificity.
The method utilizes transparent juvenile fish of zebra fish to observe the movement of cells in real time, and the process of cell movement can be completely recorded through software, so that the singleness of in vitro experiments is compensated, the in vivo real-time observation covers the in vivo complex regulation and control relationship, and the change of cell functions caused by genetic variation can be more accurately estimated;
the method is based on animal models to identify the functional variation of genetic variation, does not depend on patient samples, is convenient for data acquisition and storage, and has high experimental repeatability and accuracy.
Through the evaluation of the invention, the potential of p.R354X for developing immune diseases (such as infectious diseases or autoimmune diseases including chronic infection, diabetes or rheumatoid arthritis and the like) caused by influencing the impaired migration function of neutrophils is suggested.
Drawings
FIG. 1 is a schematic representation of zebra fish elmo1 gene deletion mutants and their sibling wild-type DNA sequencing results and their corresponding formed proteins.
FIG. 2 is a schematic representation of phenotypic analysis of zebra fish elmo1 gene deletion mutants and their sibling wild type (elmo1+/+), heterozygous (elmo+/-) zebra fish neutrophils.
FIG. 3 is a schematic representation of the results of rescue experiments in which the zebra fish elmo1 gene was specifically expressed in neutrophils in zebra fish elmo1 gene deletion mutants (elmo 1-/-) and sibling zebra fish thereof (elmo1+/+ or elmo+/-).
FIG. 4 is a schematic representation of the results of rescue experiments in zebra fish ELMO1 gene deletion mutants (ELMO 1-/-) and their sibling zebra fish (elmo1+/+ or elmo+/-) specifically expressing human ELMO1 gene and its genetic variation sites in neutrophils.
FIG. 5 is a graph of the movement trace of neutrophils on the yolk sac at 3dpf of mutant zebra fish and sibling zebra fish.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the examples below, unless otherwise specified, are all conventional.
The term "wild-type" or "WT" as used herein refers to wild-type zebra fish.
The term "sibling" as used herein refers to the production of a polypeptide from a offspring individual of the same parent, either sibling wild type or sibling heterozygous (elmo 1 +/+ Or elmo +/- )。
The in vivo imaging techniques in the following examples were performed by time-lapse in vivo imaging with reference to methods reported in the literature (Xu, j., wang, t., wu, y., jin, w., and Wen, z. (2016) Microglia Colonization of Developing Zebrafish Midbrain Is Promoted by Apoptotic Neuron and lysophosphotidyline.dev Cell 38, 214-222).
Specifically, larvae 3 days after fertilization (3 dpf) were anesthetized in 0.01% tricaine (A5040; sigma-Aldrich), the anesthetized larvae were then fixed in 1% low melting agarose, and the larvae were imaged on a Zeiss 880 confocal microscope with a constant temperature and humidity incubator at 28 ℃. We used a 10-fold objective for neutrophil tracking. The Z step is set to 3 millimeters and typically 15-20 planes are acquired in the Z-plane at intervals of less than 3 minutes. The captured images were image processed using ImageJ software and cell tracking analysis using MTrackJ plug-in. The tracking paths of the single cells are extracted from the derived tracking results and are combined in layers using Photoshop software. The derived cell movement velocity data was statistically analyzed using statistical software Prism 7.
Examples
1. Zebra fish culture
Zebra fish cultures were grown as reported in The literature (Westerfield M: the zebrafish: guide for The laboratory use of zebrafish (Brachdanio rerio). Edition by Eugene, OR, M.Westerfield, 1993).
The following zebra fish strains were used in the present invention:
ABSR wild type zebra fish, transgenic line Tg (lyz: dsRed), elmo1 gene deleted zebra fish mutant Tg (lyz: dsRed): from the zing Wen laboratories (L.Li, B.Yan, Y.Q.Shi, W.Q.Zhang, Z.L.Wen, live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration.j Biol Chem 287,25353-25360 (2012)).
The elmo1 gene deletion zebra fish mutant utilizes a TALEN gene editing technology to design a target site on exon 18 of the elmo1 gene (NC_ 007130.7). Using this technique, 13 base pairs (GCCCTCAGGGAGA, SEQ ID NO. 1) were knocked out on the PH domain where the zebra fish Elmo1 protein and the Dock2 protein bind. The deletion of base pairs causes frame shift mutation, which leads to premature stop codons, leads to the loss of PH structural domains and finally generates gene deletion mutants of the zebra fish Elmo1 protein, which are nonfunctional.
FIG. 1 is a schematic representation of a zebra fish elmo1 gene deletion mutant and its sibling wild-type DNA sequencing results and its corresponding formed protein, wherein, FIG. A represents a schematic representation of elmo1 genomic locus (NC_ 007130.7). The extended region on exon 18 represents the sequence targeted by the TALEN system. The left light grey sequence indicates TALEN arm binding sites. The light grey sequence on the right represents the TALEN arm binding site. The middle dark sequence is a spacing site. elmo1 +/+ Corresponds to the sibling wild type, whereas elmo1 -/- Representing a loss-of-function mutant. Dashes represent 13 base pair deletions. Panel B represents a schematic representation of the wild type (Elmo 1 wt) and mutant Elmo1 proteins (Elmo 1 mut). Of these, the Elmo1 wt of 726 amino acids (aa) contains five conserved domains (np_ 998256), whereas Elmo1 mut results in a truncated protein ending at 619 aa. RBD represents RhoG protein binding domain; EID stands for ELMO inhibition domain; ELMO1 represents an ELMO1 domain; PH represents the pleckstrin homology domain; EAD stands for ELMO autoregulation domain.
2. Establishment of gene mutation site expression system
2.1 establishment of an expression System for specific expression of the zebra fish elmo1 Gene by neutrophils
For specific expression of the zebra fish Elmo1 gene in neutrophils, we have reported in the literature that a lyz promoter was used and that the zebra fish Elmo1 gene coding region (NM-213091.1) was ligated after lyz promoter and that the P2A self-cleaving peptide was used to ligate the zebra fish Elmo1 protein with the green fluorescent protein GFP. We cloned the above components into the vector PBLK-sv40 plasmid. The final plasmid obtained: PBLK-lyz elmo1 ze P2A-GFP-sv40 by microinjection, 1.8nl (40 ng/. Mu.l) of plasmid was injected into the single cell stage elmo1 gene deletion mutant and the sibling zebra fish embryo, and the zebra fish embryo was cultured to 3 days after birth (3 dpf) and a time-lapse in vivo imaging experiment was performed to analyze whether the zebra fish elmo1 gene regulated the neutrophil migration function through the cell autonomy mechanism.
FIG. 2 shows a zebra fish elmo1 gene deletion mutant and a sibling wild type thereof (elmo 1 +/+ ) Heterozygous (elmo) +/- ) ZebraPhenotypic analysis of fish neutrophils. Wherein Panel A is sibling wild type (elmo 1 +/+ ) And mutant (elmo 1) -/- ) In situ hybridization results of zebra fish. Panel B shows neutrophil signaling number, as shown in panels A and B, compared to the sibling fish, mutant (elmo 1 -/- ) Is not significantly altered. Panel C shows the movement trace of neutrophils marked by Tg (lyz: dsRed) on the yolk sac at 3dpf of the sibling wild type, heterozygous and mutant zebra fish. Wherein each successive line represents the trajectory of movement of each different neutrophil. Panel D is a statistical result of neutrophil migration function, and shows that compared with the wild type and heterozygous zebra fish of the sibling, the migration speed of the neutrophil in the mutant zebra fish is obviously reduced. (one-way analysis of variance, ns: no significant differences.)
A rescue experiment was then performed, i.e., zebra fish elmo1 gene was expressed in the elmo1 gene deletion mutant, and the results are shown in FIG. 3.
FIG. 3 shows the deletion of the elmo1 gene in zebra fish (elmo 1 -/- ) And the sibling zebra fish (elmo 1) +/+ Or elmo +/- ) Results of rescue experiments in which the zebra fish elmo1 gene was expressed specifically in neutrophils. Wherein, panel A shows that when mutant zebra fish and sibling zebra fish are 3dpf, they are Tg on the yolk sac (lyz:elmo 1 ze -P2A-GFP) labeled neutrophil movement trajectories. Wherein each successive line represents the trajectory of movement of each different neutrophil. Panel B is a statistical analysis of neutrophil migration function, showing that, with injection of control plasmid lyz: compared with the zebra fish of GFP, after the zebra fish elmo1 gene is expressed, the migration capacity of neutrophils in the mutant is obviously recovered. (one-way analysis of variance, ns: no significant difference, p<0.001。)
This result demonstrates that the damaged neutrophil migration function is restored after the elmo1 gene of the zebra fish is expressed by the elmo1 gene deletion mutant, and proves that the zebra fish elmo1 gene regulates the neutrophil migration function through a cell autonomy mechanism.
2.2 establishment of an expression System for specific expression of the human ELMO1 Gene by neutrophils
For the specific expression of the human ELMO1 gene in neutrophils, we have reported in the literature that, as mentioned previously, the lyz promoter was chosen. And the human ELMO1 gene coding region (NM_ 014800.11) is connected behind the lyz promoter, and the human ELMO1 protein and green fluorescent protein GFP are directly connected to obtain the ELMO1-GFP fusion protein. We cloned the above components into the vector PBLK-sv40 plasmid. The final plasmid obtained: PBLK-lyz ELMO1 hu GFP-sv40 (hu-WT) by microinjection, 1.8nl (40 ng/. Mu.l) of plasmid was injected into the single cell stage Elmo1 gene deletion mutant and the sibling zebra fish embryo, and the zebra fish embryo was cultured to 3 days after birth (3 dpf) and a time-lapse in vivo imaging experiment was performed to confirm whether the human Elmo1 gene and the zebra fish Elmo1 gene have high conservation to the regulation of neutrophil migration function.
As shown in FIG. 4C, after the ELMO1 gene deletion mutant expresses the human ELMO1 gene, the damaged neutrophil migration function is recovered, which proves the high conservation of the human ELMO1 gene and the zebra fish ELMO1 gene to the regulation of the neutrophil migration function. This result demonstrates that the use of zebra fish elmo1 gene mutants as pathogenic animal models for assessing expression of the site of genetic variation is feasible.
2.3 establishment of expression System for specific expression of human ELMO1 Gene mutation sites by neutrophils
(1) Screening human ELMO1 gene mutation sites.
ELMO1 gene mutation site information is searched from a clinical genetic database established by the early detection sample of the inventor company, and the searched mutation site screening basis is as follows: the Mutation is annotated by adopting VEP (variant Effect Predictor) software, mutation sites are screened on the basis of ClinVar, OMIM, HGMD genetic disease databases such as gnomAD, mutation databases and crowd large-scale sequencing databases such as thousand genome and ESP6500, and Mutation pathogenicity is predicted and classified by adopting various computer algorithms (PolyPhen 2, SIFT, mutation Taster and the like).
The classification of variations is categorized with reference to the sequence variation interpretation standards and guidelines issued by the american society of medical genetics (ACMG). From among them, 14 ELMO1 gene mutation sites classified as VUS were selected, as shown in Table 1 below, in which the 11-14 gene mutation sites were not conserved in humans and zebra fish, and were not involved in the present invention. According to whether the nature of the amino acid is changed after the basic group mutation of the gene, the mutation site of the gene 1-3 is reserved for functional verification in the invention.
TABLE 1 ELMO1 Gene mutation site information
(2) The human ELMO1 gene mutation site is specifically expressed in neutrophils.
For specific expression of the human ELMO1 gene mutation site in neutrophils, as mentioned above, the lyz promoter was chosen. After base editing, the lyz promoter was ligated with the human ELMO1 gene mutation sites No. 1-3 (p.E90K, p.D194G and p.R354X), and the human ELMO1 mutation protein and the green fluorescent protein GFP were directly ligated to obtain an ELMO1-GFP mutation fusion protein.
We cloned the above components into the vector PBLK-sv40 plasmid. The final plasmid obtained: PBLK-lyz ELMO1 VUS GFP-sv40 (p.E90K, p.D194G and p.R354X) by microinjection, 1.8nl (40 ng/. Mu.l) of plasmid was injected into single cell stage ELMO1 gene deletion mutant and the sibling zebra fish embryo, and the zebra fish embryo was cultured to 3 days postnatal (3 dpf) for a time-lapse in vivo imaging experiment to analyze pathogenicity of human ELMO1 gene mutation site.
The results are shown in FIGS. 4-5, and FIG. 4 shows the result of deletion of the elmo1 gene in zebra fish (elmo 1 -/- ) And the sibling zebra fish (elmo 1) +/+ Or elmo +/- ) A rescue experiment of expressing human ELMO1 genes and gene mutation sites thereof in neutrophils. Panel A shows a schematic protein representation of the human ELMO1 gene and marks on the map the locus of three genetic variations according to the present inventionFunctional domain location. Panels B and C are statistical results of neutrophil migration function. Figure 5 shows the movement trace of neutrophils on the yolk sac at 3dpf of mutant zebra fish and sibling zebra fish. Panel B shows that Tg (lyz: ELMO 1) was injected bearing the coding region of the human ELMO1 gene hu-WT -GFP) plasmid-derived neutrophils move within the zebra fish elmo1 mutant and siblings, respectively. Panels C-E show the movement trajectories of neutrophils in the zebra fish ELMO1 mutant and its siblings, respectively, after expression of three ELMO1 gene mutation sites, p.e90k, p.d194g and p.r4x, respectively, with each continuous line representing the movement trajectory of each different neutrophil.
The results show that after p.E90K and p.D194G are expressed, the migration function of neutrophils in the mutant is recovered, while after p.R354X is expressed, the migration function of the neutrophils of the mutant cannot be recovered, and the migration function of the neutrophils of the zebra fish is inhibited. The statistical method in the figure adopts single-factor analysis of variance, ns: no significant difference, p <0.001.
After expressing the human ELMO1 gene mutation sites, p.E90K and p.D194G can both recover the damaged neutrophil migration function in the zebra fish mutant, which indicates that the two gene mutation sites have no strong obvious pathogenicity. But p.R354X cannot restore the damaged neutrophil migration function in the mutant zebra fish, and besides, the normal neutrophil migration function in the mutant zebra fish is influenced, so that the mutant zebra fish has obvious pathogenicity, namely has pathological potential and can be related to the occurrence and development of chronic inflammation.
In summary, in the zebra fish elmo1 gene deletion mutant, the migration function of the neutrophils was impaired compared to the sibling wild-type and heterozygous zebra fish. And after the zebra fish elmo1 gene is expressed, the migration function of the damaged neutrophil in the mutant body is recovered. The human ELMO1 gene expression can also restore the damaged neutrophil migration function in the mutant body of the zebra fish, thus proving the high conservation of human and zebra fish. Therefore, after the human ELMO1 gene mutation sites are expressed, p.E90K and p.D194G can restore the damaged neutrophil migration function in the zebra fish mutant body, and the two gene mutation sites are not strong and obvious in pathogenicity. But p.R354X cannot recover the damaged neutrophil migration function in the zebra fish mutant, and in addition, the normal neutrophil migration function in the sibling fish is also influenced, which indicates that the zebra fish mutant has obvious pathogenicity, namely has pathological potential.
The ELMO1 gene variant protein is transiently over-expressed in the zebra fish ELMO1 gene mutant, so that accurate assessment of the cell motility of the gene variation can be realized.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Guangzhou gold Domain medical test center Co., ltd
South China University of Technology
<120> method for evaluating pathogenicity of genetic variation by mutant cell migration function and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gccctcaggg aga 13

Claims (3)

1. A method for evaluating pathogenicity of genetic variation by mutant cell migration function, characterized by:
constructing an expression system for specifically expressing human ELMO1 gene mutation sites by neutrophils: obtaining a plasmid for expressing fusion protein of human ELMO1 mutant protein and fluorescent protein GFP after a neutrophil specific promoter lyz, cloning the plasmid carrying a gene mutation site sequence to be evaluated into an ELMO1 gene deletion zebra fish mutant model by a microinjection method, and specifically expressing the gene mutation site to be evaluated in neutrophil under the drive of the neutrophil specific promoter lyz to obtain a zebra fish gene mutation site expression system, wherein the ELMO1 gene deletion zebra fish mutant model is a zebra fish with a sequence shown as SEQ ID NO.1 knocked out by adopting a TALEN gene editing technology, and the gene mutation site to be evaluated is: the mutation site is a site corresponding to the coding region of the NM_213091.1 gene, and is p.E90K, p.D194G or p.R354X; the specific method for obtaining the zebra fish gene mutation site expression system by microinjection comprises the following steps: injecting plasmids into ELMO1 gene deletion zebra fish mutants and sibling zebra fish embryos in a single cell period, and culturing the zebra fish embryos to 3 days after birth, so that a time-lapse living imaging experiment can be performed;
time-lapse in vivo imaging: and carrying out visual tracking on the neutrophils carrying the ELMO1 gene mutation sites in the zebra fish gene deletion mutant model through time-lapse live imaging, and recording the migration path and migration speed of the neutrophils expressing the human ELMO1 gene mutation sites on the zebra fish yolk sac through the movement record of the neutrophils, and evaluating the influence of the gene mutation sites on the migration function of the neutrophils so as to evaluate the pathogenicity of the gene mutation sites.
2. The method of claim 1, wherein the migration function of neutrophils in the ELMO1 gene-deleted zebra fish mutant model is determined by expression of human ELMO1 gene mutation sites.
3. Use of the method of any one of claims 1-2 for assessing pathogenicity of genetic variation with mutant cell migration function for studying pathogenicity of human ELMO1 gene variation site; the pathogenicity is that the neutrophil movement function is damaged or enhanced, thereby increasing the susceptibility of infectious diseases or autoimmune diseases; the infectious disease or autoimmune disease is chronic infection, inflammatory enteritis or rheumatoid arthritis.
CN202110779589.XA 2021-07-09 2021-07-09 Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application Active CN113528570B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110779589.XA CN113528570B (en) 2021-07-09 2021-07-09 Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110779589.XA CN113528570B (en) 2021-07-09 2021-07-09 Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application

Publications (2)

Publication Number Publication Date
CN113528570A CN113528570A (en) 2021-10-22
CN113528570B true CN113528570B (en) 2023-08-01

Family

ID=78098304

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110779589.XA Active CN113528570B (en) 2021-07-09 2021-07-09 Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application

Country Status (1)

Country Link
CN (1) CN113528570B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114496072A (en) * 2022-01-17 2022-05-13 北京安琪尔基因医学科技有限公司 Deafness pathogenic analysis grade classification method and device, computer readable storage medium and server

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3568522B2 (en) * 2002-11-26 2004-09-22 独立行政法人 科学技術振興機構 DOCK2 functional domains and associated molecules essential for lymphocyte migration
CN111264467B (en) * 2018-12-05 2021-05-14 华南理工大学 Application of mutant zebra fish in preparation of animal model of myelodysplastic syndrome

Also Published As

Publication number Publication date
CN113528570A (en) 2021-10-22

Similar Documents

Publication Publication Date Title
Werner et al. Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke
Tessadori et al. Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders
Loria et al. A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans
Wenick et al. Genomic cis-regulatory architecture and trans-acting regulators of a single interneuron-specific gene battery in C. elegans
Naert et al. CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs
Unlu et al. Phenome-based approach identifies RIC1-linked Mendelian syndrome through zebrafish models, biobank associations and clinical studies
CN105442052A (en) Deoxyribonucleic acid (DNA) library for detecting disease causing genes of aoreic dissection diseases and application thereof
Moriya-Ito et al. Evolution of vomeronasal receptor 1 (V1R) genes in the common marmoset (Callithrix jacchus)
CN113528570B (en) Method for evaluating pathogenicity of genetic variation by using mutant cell migration function and application
Wang et al. Resequencing and SNP discovery of Amur ide (Leucis c us waleckii) provides insights into local adaptations to extreme environments
Kheirallah et al. Translating lung function genome-wide association study (GWAS) findings: new insights for lung biology
Willet et al. Canine disorder mirrors human disease: exonic deletion in HES7 causes autosomal recessive spondylocostal dysostosis in miniature Schnauzer dogs
Barros-Becker et al. Distinct tissue damage and microbial cues drive neutrophil and macrophage recruitment to thermal injury
Lynch et al. Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal
CN104040337A (en) Screening method
Belele et al. Differential requirement for Gata1 DNA binding and transactivation between primitive and definitive stages of hematopoiesis in zebrafish
Jasinska Resources for functional genomic studies of health and development in nonhuman primates
Morgan et al. Molecular evolution of mammalian genes with epistatic interactions in fertilization
Zhang et al. Tnni1b-ECR183-d2, an 87 bp cardiac enhancer of zebrafish
Verhagen et al. Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy
EA006367B1 (en) Schizophrenia-related voltage-gated ion channel gene and protein
Sayer et al. Interaction of the nuclear matrix protein NAKAP with HypA and huntingtin: implications for nuclear toxicity in Huntington’s disease pathogenesis
Xue et al. Functional verification of novel ELMO1 variants by live imaging in Zebrafish
Diri et al. The nucleome of developing murine rod photoreceptors
Klämbt et al. Genetic variants in ARHGEF6 cause congenital anomalies of the kidneys and urinary tract in humans, mice, and Frogs

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information
CB02 Change of applicant information

Address after: No. 10, Helix 3 Road, International Biological Island, Huangpu District, Guangzhou City, Guangdong Province, 510320

Applicant after: GUANGZHOU KINGMED CENTER FOR CLINICAL LABORATORY

Applicant after: SOUTH CHINA University OF TECHNOLOGY

Address before: 510335 3rd floor, 2429 Xingang East Road, Haizhu District, Guangzhou City, Guangdong Province

Applicant before: GUANGZHOU KINGMED CENTER FOR CLINICAL LABORATORY

Applicant before: SOUTH CHINA University OF TECHNOLOGY

GR01 Patent grant
GR01 Patent grant