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Preparation method and application of transgenic zebra fish capable of specifically removing T cells

A zebrafish and transgenic technology, applied in the field of zebrafish model preparation, can solve the problems of stunted growth and development, low embryo survival rate, high requirements on breeding environment, etc.

Active Publication Date: 2020-10-30
NANJING UNIV OF TECH
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  • Abstract
  • Description
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Problems solved by technology

However, these zebrafish strains have higher requirements on the feeding environment due to immunodeficiency, and also have growth retardation and low embryo survival rate.

Method used

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  • Preparation method and application of transgenic zebra fish capable of specifically removing T cells
  • Preparation method and application of transgenic zebra fish capable of specifically removing T cells
  • Preparation method and application of transgenic zebra fish capable of specifically removing T cells

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Embodiment 1

[0040] 1. Experimental materials and methods

[0041] 1. Experimental animals

[0042] According to the standardized protocol (see: Westerfield, M. The zebrafish book: A guide for the laboratory use of zebrafish (Danio Rerio) 1989, Eugene, Oregon: University of Oregon Press.), breed wild-type zebrafish (AB strain), water temperature 28.5 ℃ , the light / dark cycle is 14h / 10h, the adult zebrafish embryos are collected after spawning, cultured in E3 hatching solution, and the number of hours post-fertilization (hpf) or days post-fertilization (dpf) Denotes developmental stages of embryos and larvae.

[0043] 2. Preparation of transgenic zebrafish

[0044] Transgenic zebrafish Tg(lck: NTR-mvenus) was prepared by Tol2 system.

[0045] 2.1 Construction of pTol2-lck-NTR-mVenus recombinant plasmid

[0046] 2.1.1 Amplify Escherichia coli NTR gene by PCR technique, its sequence is shown in SEQ ID NO.2, and its primer sequence is: forward primer: 5'-AGGCGGCCGCATGGATATCATTTCTGTCG-3' (S...

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Abstract

The invention discloses a preparation method and application of transgenic zebra fish capable of specifically eliminating T cells. The preparation method comprises the following steps: introducing recombinant plasmids pTol2-lck-NTR-mVenus and Tol2 transposase mRNA into a wild zebra fish embryo together, and culturing to obtain the stably inherited transgenic zebra fish strain, wherein the stably inherited transgenic zebra fish line is a transgenic zebra fish capable of specifically eliminating T cells, and the recombinant plasmid pTol2-lck-NTR-mVenus carries a zebra fish lck gene promoter sequence and an escherichia coli NTR gene sequence. By utilizing the method, the transgenic zebrafish which specifically expresses the NTR gene in the T cell and can be stably inherited can be effectivelyprepared, in addition, under the action of metronidazole, few T cells of the transgenic zebra fish can be specifically removed, so that the transgenic zebra fish has immunologic function defects, andfurthermore, the transgenic zebra fish can provide an animal model for living body research on immune cell development, organ transplantation, tumor xenograft and related drug screening.

Description

technical field [0001] The invention relates to a preparation method and application of a zebrafish model, in particular to a transgenic zebrafish capable of specifically eliminating T cells and a preparation method and application thereof. More specifically, the present invention relates to a method for preparing a transgenic zebrafish that can specifically clear T cells, a transgenic zebrafish that can specifically clear T cells and its research on immune cell development, research on organ transplantation, and research on tumor xenografts and related drug screening applications. Background technique [0002] Zebrafish tumor xenotransplantation has become an innovative model for studying tumors in recent years due to its small size, low breeding cost, short sexual maturation cycle, in vitro development and transparent embryos, which are convenient for in vivo observation. At present, zebrafish tumor xenotransplantation mostly selects newly hatched juvenile fish, and human...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/87C12N15/65A01K67/027
CPCC07K14/245C12N9/1241C12N15/8509C12N15/87C12N15/65A01K67/0278A01K2227/40A01K2267/03A01K2267/025C12N2015/8527Y02A40/81
Inventor 李崇勇何明芳蔡阳
Owner NANJING UNIV OF TECH
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