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Preparation method of transgenic zebrafish

A zebrafish and transgenic technology, applied in the field of genetic engineering, can solve problems such as strong toxicity, carcinogenesis, and endocrine disruption

Inactive Publication Date: 2017-08-18
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This type of compound is extremely toxic, extremely difficult to degrade in the natural environment, can migrate long distances around the world, is not easy to decompose after being ingested by organisms, and is gradually concentrated and enlarged along the food chain. It has carcinogenic, teratogenic, endocrine disruption

Method used

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  • Preparation method of transgenic zebrafish
  • Preparation method of transgenic zebrafish
  • Preparation method of transgenic zebrafish

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Embodiment Construction

[0065] The specific steps of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0066] Step 1: Transformation of CYP1A promoter sequence

[0067] 1. Transformation of CYP1A promoter containing 6 DREs

[0068] 1. Design a pair of primers CYP1A-F and CYP1A-R using the zebrafish genome sequence known on the National Center for Biotechnology Information (NCBI) website, and clone the CYP1A (Chromosome 18) promoter sequence. The primer sequences are as follows:

[0069] CYP1A-F: 5'-AAA CTCGAG GAAACCCACGCCATGCAAAC-3' (add XhoI restriction site)

[0070] CYP1A-R: 5'-AAA GGATCC GCTGCACTATTCCAGCGGTA-3' (Add HindⅢ restriction site)

[0071] 2, use Genomic DNA Kit was used to extract zebrafish genomic DNA, and the PCR components were mixed according to the following system: 50μL system

[0072]

[0073]

[0074] PCR reaction conditions:

[0075]

[0076] After the amplification reaction, 2% agarose gel electrophoresis ...

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Abstract

The invention relates to a preparation method of transgenic zebrafish, and relates to gene engineering. The preparation method comprises the following steps of (1) modifying a CYPIA starter sequence; (2) establishing a Tol2 transposon system knocking plasmid, and preparing a microinjection sample; (3) performing microinjection and subculture on zebrafish embryos. The invention provides an establishing method of a CYP1A starter containing twelve DREs (dehydration-responsive element), and a recombination Tol2 transposon knocking plasmid which contains the starter and is connected with a green fluorescent protein gene, a plasmid linear purifying method, and a microinjection sample method. The Tg(pCYP1A-6*DRE-EGFP) and Tg(pCYP1A-12*DRE-EGFP) transgenic zebrafish prepared by the microinjection technique can be used for comparing the activities of the starters respectively containing six DREs and twelve DREs.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a method for preparing transgenic zebrafish. Background technique [0002] Persistent organic pollutants (POPs) refer to long-distance migration and long-term existence in the environment through various environmental media (atmosphere, water, organisms, etc.), with long-term persistence, bioaccumulation, semi-volatile and highly toxic, natural or synthetic organic pollutants that pose serious hazards to human health and the environment. [0003] Dioxins are a typical class of persistent organic pollutants, which exist in the natural environment for a long time and have attracted great attention. This type of compound is extremely toxic, extremely difficult to degrade in the natural environment, can migrate long distances around the world, is not easy to decompose after being ingested by organisms, and is gradually concentrated and enlarged along the food chain. It has carcinogenic, terato...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCC12N15/8509A01K67/0278A01K2217/203A01K2227/40A01K2267/0393C12N2800/60
Inventor 左正宏沈超周懿翕王重刚
Owner XIAMEN UNIV
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