Preparation of IL7R gene-deleted zebra fish mutant and application thereof

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: 2017-08-18
NANKAI UNIV
View PDF3 Cites 43 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation of IL7R gene-deleted zebra fish mutant and application thereof
  • Preparation of IL7R gene-deleted zebra fish mutant and application thereof
  • Preparation of IL7R gene-deleted zebra fish mutant and application thereof

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/85A01K67/027C12Q1/68
CPCA01K67/0276A01K2217/075A01K2227/40C07K14/461C12N15/1138C12N15/8509C12N2310/10C12N2800/106C12N2800/80
Inventor 李玉皓蔡世娇陈阳
Owner NANKAI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap