Supercharge Your Innovation With Domain-Expert AI Agents!

Method and application of cultivating transgenic zebrafish labeled central nervous system oligodendrocytes

A zebrafish and gene technology, applied in the field of special plasmid for transgenic fish, zebrafish lingo-1 gene promoter, special plasmid for cultivating lingo-1 transgenic zebrafish, cultivating lingo-1 transgenic zebrafish, can solve exogenous low gene expression

Inactive Publication Date: 2016-08-03
UNIV OF SCI & TECH OF CHINA
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of foreign genes will be regulated by the promoter constructed in the expression vector. Since the driving strength of the promoter is affected by multiple regulatory factors, the use of heterologous promoters may lead to low expression of foreign genes

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
  • Method and application of cultivating transgenic zebrafish labeled central nervous system oligodendrocytes
  • Method and application of cultivating transgenic zebrafish labeled central nervous system oligodendrocytes
  • Method and application of cultivating transgenic zebrafish labeled central nervous system oligodendrocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Breeding lingo-1 transgenic zebrafish

[0036] (1) Construction of lingo-1 recombinant expression vector

[0037] Use HindIII and XhoI double digestion plasmid pEGFP-1 (purchased from BDBiosciencesClontech) to obtain about 1Kb EGFP fluorescent protein coding frame and SV40polyA fragment; design zebrafish lingo-1 gene promoter-specific primers (see Table 1 below) and perform genome sequencing After PCR, the target fragment was recovered and purified by double digestion with BamHI and HindIII to obtain about 2Kb lingo-1 gene promoter sequence (SEQ ID No: 1); plasmid pI-SceI-pBSII-SK+ (Genbank: DQ836146 , see Thermes, V., et al 2002, where pBSII-SK+ is available from Stratagene).

[0038] A fragment of about 3Kb was obtained; the above three fragments were connected at the same time to obtain a recombinant vector pI-Scel-lingo1-EGFP (SEQ ID No: 2).

[0039] Table 1. Zebrafish lingo-1 gene promoter-specific primers:

[0040] primer name

sequence

...

Embodiment 2

[0048] Example 2. Screening of drugs or compounds that affect the differentiation and maturation of oligodendrocytes

[0049] The AB wild type (purchased from the National Zebrafish Model Animal Center) was crossed with the lingo-1:EGFP strain obtained according to the method in Example 1 to obtain lingo-1:EGFP / AB hybrid zebrafish juveniles for screening.

[0050] For example, using the Prestwick Chemical Library (Prestwick-Chemical), an existing chemical synthesis library, about a thousand compounds are selected for preliminary screening (laboratory small-scale screening). The selected compounds were prepared with a final concentration of 10uM compound working solution with 10mM DMSO, and 3 strips of 30hpflingo-1:EGFP / AB juveniles / well (9 strips for each compound) and 200ul compound working solution / well were placed in 96 well plates and raised to 72hpf. First, the toxicity was detected. The juvenile fish in the compound group with no toxicity was fixed with 4% PFA, and the ...

Embodiment 3

[0052] Example 3. Screening of drugs or compounds that affect oligodendrocyte remyelination

[0053] The AB wild-type and lingo-1:EGFP lines were crossed to obtain lingo-1:EGFP / AB heterozygous adult zebrafish (>6 mpf) for screening. A fish optic nerve demyelination model (No. 5 forceps CRUSH or optic nerve microinjection LPC) was constructed, and the compounds to be screened were dissolved and absorbed into a medicinal sponge and wrapped around the optic nerve. Seven days after the operation, the adult fish were anesthetized and the optic nerve was removed, fixed with 4% PFA, frozen sectioned, and immunohistochemically studied for oligodendrocytes (EGFP+) and myelination (using zebrafish MBP antibody, synthesized by Abmart) .

[0054]Those skilled in the art should understand that high-throughput screening can be performed by using the lingo-1:EGFP / AB hybrid zebrafish adult screening model according to the above operation steps, and drugs that affect oligodendrocyte remyelina...

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 a method for cultivating transgenic zebra fish marking the oligodendrocyte of a central nervous system, an application of the transgenic zebra fish obtained by the method and special plasmid thereof. The method for cultivating transgenic zebra fish comprises the following steps: (1) establishing a lingo-1 recombinant expression vector, wherein the lingo-1 recombinant expression vector comprises an appropriate endonuclease digestion site, a zebra fish lingo-1 gene promoter sequence, a fluorescent protein coding frame and a poly A fragment; (2) converting the zebra fish zygote with the recombinant expression vector in the step (1) to obtain an F0 generation; (3) hybridizing the F0 generation with wild zebra fish to obtain the transgenic zebra fish marked by the lingo-1 specific gene. In the method disclosed by the invention, the zebra fish lingo-1 gene promoter is also identified, and the nucleotide sequence is SEQ ID No:1.

Description

technical field [0001] The invention relates to a method for cultivating transgenic fish marked with oligodendrocytes of the central nervous system and the application of the transgenic fish obtained by the method. Special plasmid for transgenic fish. Specifically, the present invention relates to a method for cultivating lingo-1 transgenic zebrafish, the application of the lingo-1 transgenic zebrafish obtained by the method, and a special plasmid for cultivating lingo-1 transgenic zebrafish. The present invention also identifies the zebrafish lingo-1 gene promoter, whose nucleotide sequence is SEQ ID No: 1. Background technique [0002] Myelination is the process by which glial cells recognize and attach to appropriate axons to form layers of tightly packed myelin sheaths (Simons, 2006; Frankline et al., 2008). The myelination process in many vertebrates has been extensively studied. The function of myelin is to increase the conduction velocity of nerve impulses and main...

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 Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10A01K67/033A61K49/00
Inventor 殷梧胡兵
Owner UNIV OF SCI & TECH OF CHINA
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com