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Preparation and application of il7r gene deletion zebrafish mutant

A gene deletion, zebrafish technology, applied in the field of molecular biology, to achieve the effect of convenient in vivo experimental research

Active Publication Date: 2019-08-02
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zebrafish IL7R is 73% similar to human IL7R. It is an effective way to use IL7R gene knockout mutants for research. However, there is no report at home and abroad.

Method used

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  • Preparation and application of il7r gene deletion zebrafish mutant
  • Preparation and application of il7r gene deletion zebrafish mutant
  • Preparation and application of il7r gene deletion zebrafish mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Screening for differentially expressed genes in a demyelination zebrafish model

[0049] 1. Steps

[0050] Using a gene chip (Affymetrix GeneChip Zeb Gene 1.0ST Array), the gene expression profile of the demyelination zebrafish model (the construction method is the same as the patent document: ZL2014101092570) was detected and analyzed. The criteria for judging differentially expressed genes are: ≥2 times is up-regulated; ≤0.5 times is down-regulated. Chip analysis found 1364 differential genes, including 271 up-regulated genes and 1093 down-regulated genes ( Figure 4 ). Among the many down-regulated genes, it was found that the expression of IL7R was significantly down-regulated (foldchange=0.44, *p=0.0061888). Real-time fluorescent quantitative PCR (qRT-PCR) and Western blot (Westernblot, WB) were used for verification. The verification method is: the experimental group is 5dpf Tg(mbp:nfsB-egfp), treated with 0.2%DMSO-5mM metronidazole (mitronidazole, M...

Embodiment 2

[0051] Example 2 Screening of sgRNA

[0052] 1. Experimental animals

[0053] Wild-type zebrafish (TU strain) was cultured according to a standardized protocol, the water temperature was 28.5°C, and the light / dark cycle was 14h / 10h. Embryos were collected after spawning of adult zebrafish and cultured in E3 hatching solution. The number of hours of fertilization (hours post- Fertilization, hpf) or days post-fertilization (dpf) indicate the developmental stage of embryos and larvae.

[0054] 2. CRISPR / Cas9 gene knockout target site design

[0055] Query the genomic DNA sequence and functional domain of the zebrafish IL7R gene at http: / / asia.ensembl.org, and design a pair of IL7R at http: / / crispr.mit.edu / according to the principle of CRISPR / Cas9 knockout The target site of the gene, the target site contains 20 bases, the selection criteria of the target site is: 5'-GG-(N)20-NGG-3'; the GG dinucleotide at the 5' end is the T7 promoter The 3' end of the target site is NGG (N i...

Embodiment 3

[0063] Example 3 Microinjection of zebrafish embryos and determination of target site effectiveness

[0064] 1. sgRNA synthesis in vitro

[0065] 1.1PCR method to obtain the template for in vitro transcription of sgRNA

[0066] The forward primer sequence is: 5'-TAATACGACTCACTATAGACTCCACTCACTCCAGTCACgttttagagctagaaatagc-3' (the first 18 nucleotide sequences are the T7 promoter, the 19-38 nucleotide sequence is the sgRNA target sequence, and the nucleotide sequence represented by lowercase letters is the sgRNA upstream backbone); The reverse primer sequence is the downstream backbone of 25bp sgRNA, the sequence is 5'-AAAAAAAGCACCGACTCGGTGCCAC-3', and the amplification is carried out according to the following PCR conditions: pre-denaturation at 95°C for 3min; denaturation at 95°C for 30s, annealing at 54°C for 30s, extension at 72°C for 20s for 35 cycle, then 10min at 72°C;

[0067] 1.2 PCR product recovery and purification;

[0068] 1.3 RNA in vitro transcription kit (MAXIs...

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Abstract

The invention discloses an IL7R gene-deleted zebra fish mutant and a preparation method thereof. The construction of the IL7R gene-deleted zebra fish mutant is realized through a CRISPR / Cas9 technology. Moreover, the invention also discloses application of the IL7R gene-deleted zebra fish mutant; a mutant model provided by the invention can be used for studying an effect of IL7R in the nervous system development and myelination.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a gene knockout zebrafish mutant, in particular to an IL7R gene-deleted zebrafish mutant and its preparation method and application. Background technique [0002] Interleukin 7 receptor (IL7R) is a transmembrane receptor belonging to a member of the type I cytokine receptor family. It consists of an α chain (IL7Rα, CD127) and a γ chain (γc, CD132). Specific component; γ chain is a common component of cytokine receptors such as IL2, IL4, IL7, IL9, IL15, IL21, also known as common γ chain, and is an essential component for initiating the expression of IL7R downstream signals. Under physiological conditions, IL7R is an essential factor for the proliferation and differentiation of lymphocytes; under pathological conditions, IL7R functional deficiency can lead to severe combined immunodeficiency disease, and acquired mutations of IL7R can cause acute T lymphocytic leukemia and so on. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/85A01K67/027C12Q1/6888
CPCA01K67/0276A01K2217/075A01K2227/40C07K14/461C12N15/1138C12N15/8509C12N2310/10C12N2800/106C12N2800/80
Inventor 李玉皓蔡世娇陈阳
Owner NANKAI UNIV
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