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Cultivation method of transgenic zebrafish for detecting organic pollutants in water body

A technology of organic pollutants and zebrafish, which is applied in the fields of plant genetic improvement, botany equipment and methods, biochemical equipment and methods, etc. The effect of high sensitivity and high luciferase activity

Pending Publication Date: 2022-05-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the technical problems of time-consuming and laborious biological monitoring in the prior art and cannot be used for intuitive monitoring, the present invention provides a method for cultivating transgenic zebrafish for detecting organic pollutants in water bodies

Method used

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  • Cultivation method of transgenic zebrafish for detecting organic pollutants in water body
  • Cultivation method of transgenic zebrafish for detecting organic pollutants in water body
  • Cultivation method of transgenic zebrafish for detecting organic pollutants in water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1p

[0053] Example 1 Preparation of pISceI-GAcyp1apromoter-eGFP / Luc plasmid

[0054] (1) Genomic DNA Extraction of Mosquito Fish

[0055] Mosquito fish were taken, 20-30 mg of caudal fins were cut, rinsed several times in distilled water, blotted dry with absorbent paper, placed in a 1.5 mL centrifuge tube, cut into pieces, and extracted according to tissue DNA kit (purchased from Tiangen Biochemical Technology (Beijing, China). ) Co., Ltd.) to extract the mosquito fish genome DNA.

[0056] (2) Amplification primer design

[0057] According to the sequence of the coding region of the mosquito fish cyp1a gene (Gene ID: MK286594.1) searched on NCBI, its cyp1a promoter sequence was retrieved from the mosquito fish genome database (GCA_019740435.1), and the upstream amplification primer GAcyp1a was designed -F: 5'-GAACCATAGGGAAGATTGCTGACAT-3', downstream amplification primer GAcyp1a-R: 5'-AAATAAAGTCAAGCATCATAGAATCATC-3'.

[0058] (3) PCR amplification of the mosquito fish cyp1a gen...

Embodiment 2p

[0073] Example 2 Functional identification of pISceI-GAcyp1apromoter-eGFP / Luc plasmid

[0074] 1. Experimental method

[0075] (1) Cell culture

[0076] Human liver cancer cell HepG2 was used to verify the effect of fluorescence induction.

[0077] Human hepatocellular carcinoma cells HepG2 were treated with 1×10 4 Cells / well were inoculated into 96-well cell culture plates, and cultured in high-glucose DMEM medium cultured with serum and without antibiotics.

[0078] (2) Cell transfection

[0079] After the HepG2 cells in the above-mentioned 96-well plate grow to a fusion rate of 70-80%, use the Promega ViaFectTM transfection reagent to transfer the pISceI-cyp1apromoter-eGFP / Luc plasmid (SEQ ID NO.2) and the pISceI-GAcyp1apromoter-eGFP / Luc plasmid (SEQ ID NO.4), respectively with Renilla luciferase control plasmid (from Promega dual luciferase assay kit Luciferase Assay System) were co-transfected into HepG2 cells.

[0080] The HepG2 cell that transfected pISceI-cyp1ap...

Embodiment 3

[0089] Preparation and screening of embodiment 3 transgenic zebrafish

[0090] 1. Experimental method

[0091] (1) Preparation of bodyless zebrafish

[0092] According to the 5'UTR region of the zebrafish mitfa gene (Gene ID: ENSDARG00000003732), six targeting sites for the CRISPR / CAS9 gene editing system were designed and named mitfa-1-6. The specific sequences are as follows:

[0093] mitfa-1:5'-CTTCAGCTGGCCAAGACGAC-3';

[0094] mitfa-2: 5'-CAAGAACTGACCAGTCGTCT-3';

[0095] mitfa-3: 5'-GCTCGAGTACAGTCACTACC-3';

[0096] mitfa-4:5'-CAGTCACTACCAGGTGAGAT-3';

[0097] mitfa-5 (SEQ ID NO.5): 5'-GACACAAAATGTATTTAAGG-3';

[0098] mitfa-6: 5'-ACAAAATGTATTTAAGGGGG-3'.

[0099] Among them, mitfa-5 (SEQ ID NO.5) has the highest targeting efficiency and is used as the targeting site for the final insertion mutation sequence.

[0100] Using the CRISPR / CAS9 gene editing system, through homologous recombination, a random sequence (SEQ ID NO.8) was inserted between the promoter and th...

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Abstract

The invention discloses a cultivation method of transgenic zebrafish for detecting organic pollutants in a water body. The transgenic zebrafish is prepared by using a promoter for expressing the mosquito catfish cyp1a gene, plasmids for simultaneously expressing green fluorescent protein and luciferase, and body-color-free zebrafish. In the actual water quality monitoring work, technicians in the field can qualitatively evaluate the pollution condition of organic matters in the water body by directly observing the fluorescence of the transgenic zebra fish, and also can quantitatively analyze pollutants in the water body by measuring the activity of luciferase of the transgenic zebra fish.

Description

technical field [0001] The invention relates to the field of biological monitoring, in particular to a method for cultivating transgenic zebrafish for detecting organic pollutants in water bodies. Background technique [0002] Biological monitoring refers to the process of evaluating the state of environmental quality from a biological point of view, by observing the response and impact of individual organisms, populations or communities on environmental quality and its changes, and then clarifying the nature, degree and scope of environmental pollution. Including biological population and community survey, acute and chronic toxicity test, water microbiological test and fish tissue pollutant analysis. Germany was the first country to carry out biological monitoring. As early as the beginning of the 20th century, the work of using aquatic organisms to monitor water quality has been carried out. Today, developed countries such as the European Union and the United States have e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N15/12A01K67/027C12Q1/6888
CPCC07K14/461C12N15/113C12N15/8509A01K67/0275C12Q1/6888C12Q2600/158Y02A20/20
Inventor 谢少林杨冰李思颖邹记兴周爱国冯永永
Owner SOUTH CHINA AGRI UNIV
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