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Application of transgenic zebrafish in preparation of animal model for specifically marking original erythroid hematopoietic process

A specific marker, zebrafish technology, applied in the biological field, can solve problems such as unclear role, and achieve the effect of convenient, easy and low-cost breeding

Pending Publication Date: 2022-04-15
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about the expression of cd99l2 gene in primitive erythrocytes, and its role in the development of erythrocytosis is not clear

Method used

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  • Application of transgenic zebrafish in preparation of animal model for specifically marking original erythroid hematopoietic process
  • Application of transgenic zebrafish in preparation of animal model for specifically marking original erythroid hematopoietic process
  • Application of transgenic zebrafish in preparation of animal model for specifically marking original erythroid hematopoietic process

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Experimental program
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Embodiment 1

[0039] 1. Breeding of zebrafish

[0040] The method in the breeding method reference of zebrafish among the present invention (Westerfield M:The zebrafish:guidefor the laboratory use of zebrafish (Brach danio rerio).Edition by Eugene, OR, M. Westerfield, 1993) carries out.

[0041] 2. Preparation of Digoxigenin-labeled Zebrafish cd99l2 Gene Antisense RNA Probe

[0042] According to the zebrafish cd99l2 gene (NM_194369.1) sequence provided in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), the present invention uses primer 5 software to design primers, and the designed primers are as follows:

[0043] Upstream primer: 5'-TGCTGGTAGTGAAAGGCTTGAGTGATGG-3';

[0044] Downstream primer: 5'-GAGTTTGTTGAACTTGTGGCTCCTGTGC-3'.

[0045] The size of the amplified fragment is 612bp.

[0046] The present invention utilizes the TranscriptAid T7 kit kit of Thermo Scientific Company to synthesize antisense RNA probes, and the reaction system for synthesizing antisense RNA probes in vitro is...

Embodiment 2

[0057]Example 2 Preparation of Tg (cd99l2-4.7k promoter eGFP) transgenic zebrafish

[0058] Tg (cd99l2-4.7k promoter eGFP) transgenic zebrafish refers to the 4.7k promoter composed of 4705 bp before the start codon (ATG) of cd99l2 gene (including 5'UTR439 bp) and 34 bp after the start codon to regulate transcription Translationally enhanced green fluorescent protein (eGFP) expression in transgenic zebrafish. The preparation process of Tg(cd99l2 4.7k promoter-eGFP) transgenic zebrafish is as follows: image 3 , where the left figure is a schematic diagram of the construction of the recombinant plasmid, and the right figure is the screening process of the transgenic zebrafish.

[0059] 1. Cloning of cd99l2 4.7k promoter

[0060] According to the promoter sequence of the zebrafish cd99l2 gene provided by ensemble (http: / / grch37.ensembl.org / ), the present invention uses primer 5 software to design primers, and the designed primers are as follows:

[0061] Upstream primer: 5'-ac...

Embodiment 3

[0079] In the present invention, flow cytometry (FACS) and fluorescence quantitative PCR (qRT-PCR) were used to observe and compare the expression of zebrafish cd99l2 gene in different hematopoietic lineages (erythroid, myeloid and lymphoid) and blood vessels.

[0080] 1. Flow cytometry

[0081] In the present invention, embryos of different stages obtained by selfing of adult transgenic lines zebrafish Tg (globin: dsred) and Tg (coronin1a: eGFP), Tg (rag2: dsred) and Tg (fli1a: eGFP) are used to mark each lineage of different hematopoietic stages Cells and vascular cells, grind embryos for flow cytometry detection, sort the green fluorescent protein GFP and red fluorescent protein dsred fluorescent cells in the single cell suspension through FITC-A and PE-TexasRed-A channels, respectively, for the sorted cells RNA was extracted for reverse transcription to obtain cDNA, and qRT-PCR was used to determine the expression level of cd99l2 in each lineage cell at different hematopoi...

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Abstract

The invention discloses application of transgenic zebrafish in preparation of an animal model for specifically marking an original erythroid hematopoietic process. The zebrafish cd99l2 gene is found to have a spatiotemporal expression profile consistent with the original red hematopoiesis of the zebrafish, and is only highly expressed in red blood cells in the original hematopoiesis stage of the zebrafish. According to the invention, by constructing Tg (cd99l2-4. 7k promoter eGFP) transgenic zebrafish, it is further found that when cd99l2 is used as a marker, the type of the marked cell is an immature erythroid precursor cell, and erythropoiesis is not affected. Therefore, the transgenic zebrafish can be used for preparing an animal model for specifically marking an original erythroid hematopoietic process, is used for researching the original hematopoietic process and an original erythrocyte development process, and can also be used for preparing a model for screening drugs for treating erythrocyte increase or decrease diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to the application of a transgenic zebrafish in the preparation of an animal model that specifically marks primitive erythroid hematopoiesis. Background technique [0002] Zebrafish hematopoietic development consists of two distinct but overlapping timelines: the primitive hematopoietic phase and the committed hematopoietic phase. During the rapid growth and development of the early embryo, the primitive hematopoietic stage assists the rapid oxidation of tissues to generate energy by producing red blood cells; the primitive hematopoietic stage lasts for a short time, and only primitive red blood cells and a small amount of macrophages are produced through differentiation during embryonic development. Hematopoietic stem cells (HSCs) with differentiation potential can be produced in the later stage of hematopoietic development, and hematopoietic stem cells can produce all...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65A01K67/027A61K49/00
Inventor 马宁卢荆澳黄春燕林芷茵彭昱轩唐政林宇
Owner SOUTHERN MEDICAL UNIVERSITY
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