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Method for inducing Epoa gene to knock out zebrafish embryonic homozygote hemoglobin phenotype

A zebrafish embryo and gene knockout technology, applied in the field of biomedicine, can solve the problems of ineffective survival, rare homozygous gene compensation in embryos, and limit the understanding and research of EPO, so as to achieve stable induction effect, increase precision, and improve operation. simple effect

Inactive Publication Date: 2018-09-28
THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the important and complex role of EPO, previous studies aimed at EPO gene knockout animal models could not survive effectively due to severe anemia, which limited further understanding and research on EPO
At present, the research on the homozygous gene compensation of Epoa gene knockout zebrafish embryos is very rare, and there is no related method to induce hemoglobin phenotype

Method used

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  • Method for inducing Epoa gene to knock out zebrafish embryonic homozygote hemoglobin phenotype
  • Method for inducing Epoa gene to knock out zebrafish embryonic homozygote hemoglobin phenotype
  • Method for inducing Epoa gene to knock out zebrafish embryonic homozygote hemoglobin phenotype

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Embodiment 1

[0060] This example provides a method for inducing the homozygous hemoglobin phenotype of Epoa gene knockout zebrafish embryos, which induces Epoa gene knockout zebrafish by gene silencing the Epob gene of Epoa gene knockout zebrafish embryo homozygotes Embryo homozygous hemoglobin phenotype.

[0061] The method specifically includes the following steps:

[0062] Step 1, construction of Epoa gene knockout zebrafish animal model:

[0063] The method disclosed in the Chinese patent "A Method for Constructing EPO Gene Knockout Zebrafish Animal Model, Primers, Plasmids and Preparation Method" with application publication number CN107217075A was used to construct the Epoa gene knockout zebrafish animal model.

[0064] Step 2, Obtaining a Mixed Embryo Group:

[0065] A mixed population of homozygous, heterozygous, and wild-type embryos containing the Epoa gene was obtained by crossing Epoa heterozygous adult zebrafish;

[0066] The specific process of step two is:

[0067] Step ...

Embodiment 2

[0107] This embodiment provides an experimental device for simultaneous gene sequencing and phenotype observation of zebrafish embryos, such as Figure 5 to Figure 12 As shown, the orifice plate main body 1 is included, and the orifice plate main body 1 is provided with reagent holes 2 distributed in an array along the horizontal and vertical directions. The orifice plate main body 1 is equipped with a groove-making top cover 3 and a cutting top cover 4 ;

[0108] The bottom surface of the tank-making top cover 3 is provided with first top plugs 31 corresponding to the reagent holes 2 one by one, and each first top plug 31 is provided with the same tank-making plate 32, which is located in the same longitudinal row. The groove-making plate 32 on each first top plug 31 is arranged in the same longitudinal vertical plane;

[0109] The bottom surface of the cutting top cover 4 is provided with second top plugs 41 corresponding to the reagent holes 2 one by one, each second top p...

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Abstract

The invention provides a method for inducing Epoa gene to knock out a zebrafish embryonic homozygote hemoglobin phenotype. According to the method, the Epoa gene for knocking out the zebrafish embryonic homozygote is subjected to gene silencing for inducing Epoa gene to knock out the zebrafish embryonic homozygote hemoglobin phenotype. The Epoa gene is subjected to gene silencing for inducing Epoagene to knock out the zebrafish embryonic homozygote hemoglobin phenotype. The method is simple in operation, stable and obvious in inducing effect, and gene sequencing and RT-PCR measurement of a single zebrafish embryo can be simultaneously achieved according to the method.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to zebrafish, in particular to a method for inducing the homozygous hemoglobin phenotype of Epoa gene knockout zebrafish embryos. Background technique [0002] As an emerging biological model, zebrafish is widely used in scientific research and the preparation of gene knockout models because of its transparent embryos, easy observation, low breeding costs, and easy gene editing operations. Previous studies on gene downregulation in zebrafish mostly used the corresponding gene Morpholino injection for gene downregulation RNA intervention. However, since Morpholino can only work for 5-7 days in zebrafish, more and more studies have questioned the inevitable off-target effects of Morpholino. Current research suggests using emerging gene editing technologies (such as CRISPR / Cas9) to construct gene Knockout zebrafish models for stable, long-term gene function examination and intervention. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/40A01K2267/03C07K14/505C12N15/85
Inventor 折剑青袁祖贻吴岳周博梁潇娄博文邓杨阳
Owner THE FIRST AFFILIATED HOSPITAL OF MEDICAL COLLEGE OF XIAN JIAOTONG UNIV
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