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Application of epc1epc2 mutant in construction of environment-susceptible zebrafish model

A 1. epc1-, zebrafish technology, applied in the field of water environment monitoring system, can solve the problem of late environmental biological monitoring, and achieve the effect of rapid emergency response and high sensitivity

Pending Publication Date: 2022-07-05
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research and system construction of environmental biological monitoring in my country started relatively late, and biological monitoring technology was gradually introduced in about the 1980s, and the monitoring system needs to be further strengthened and perfected

Method used

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  • Application of epc1epc2 mutant in construction of environment-susceptible zebrafish model
  • Application of epc1epc2 mutant in construction of environment-susceptible zebrafish model
  • Application of epc1epc2 mutant in construction of environment-susceptible zebrafish model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] get epc1 - / - epc2 - / - Methods for mutant zebrafish transgenic fish lines:

[0024] 1.1 Experimental materials

[0025] Wild-type zebrafish (Zebrafish, Danio rerio) and mutants were raised in the fish house of the School of Fisheries, Huazhong Agricultural University. The culture conditions were: 28.5±0.5°C water temperature, light and dark times of 14h and 10h, respectively, and fed with Artemia 2 per day. Second-rate.

[0026] 1.2 Experimental method

[0027] Selection and confirmation of zebrafish epc1 and epc2 gene gRNA target sites:

[0028] The target site of epc1gRNA was determined to be: 5'-ggtctggcagatcctcgcag-3'.

[0029] The epc2gRNA target site was determined to be: 5'-gggcgtctagcgctcgcgcg-3'.

[0030] 1.2.1 Using CRISPR / Cas9 gene editing technology to construct epc1 respectively - / - and epc2 - / - mutant

[0031] 1.2.1.1 In vitro synthesis of gRNA

[0032] The pMD19-TgRNA (Chang et al., 2013) plasmid was used as the template, and the upstream primer wi...

Embodiment 2

[0076] Example 2: Aeromonas hydrophila stress experiment

[0077] (1) Aeromonas hydrophila was inoculated with LB medium at 0.1% v / v, and placed in a 28°C incubator for 220 r / min shaker culture;

[0078] (2) After culturing for 8 hours, the concentration of Aeromonas hydrophila has reached a high level. At this moment, the concentration of Aeromonas hydrophila is detected by the coating plate method. Dilute the stock solution of Aeromonas hydrophila by 10 in sterile deionized water 6 100 μL of the diluted bacterial solution was coated on LB plates, three parallel, and cultured in a 28°C incubator, the number of single colonies was calculated the next day, and the average value was obtained to obtain the actual concentration of the bacterial solution;

[0079] The LC50 of the experimental pathogen Aeromonas hydrophila on zebrafish is 10 10 CFU / mL

[0080] (3) Dilute Aeromonas hydrophila with sterilized deionized water, adjust the concentration of Aeromonas hydrophila, and pr...

Embodiment 3

[0088] Hypoxic stress experiment

[0089] (1) The oxygen concentration of the hypoxic chamber is adjusted to 2%, the nitrogen concentration is 93%, the carbon dioxide concentration is 5%, and the temperature is adjusted to 28.5°C;

[0090] (2) Put an appropriate amount of zebrafish culture circulating water into the hypoxic chamber 12 hours in advance, and stir to make the oxygen content of the circulating water 2%;

[0091] (3) Collection of wild type, mutant (epc1 - / - , epc2 - / - and epc1 - / - epc2 - / - ) 72hpf juveniles. 3 replicates per group, 50 samples per replicate, placed in a 60mm petri dish, and the water in the petri dish was replaced with the water with an oxygen content of 2% prepared in step (2), and then the samples were placed in Hypoxic chamber; same treated fish at normal oxygen concentration as controls.

[0092] (4) Count and record the number of deaths every two hours;

[0093] (5) Analyze the statistical data and calculate the significance.

[0094] ...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to an epc1- / -epc2- / -mutant and application of the epc1- / -epc2- / -mutant in construction of an environment-susceptible zebrafish model, homozygous mutant zebrafish with deletion of epc1 and epc2 single genes is constructed respectively, then the homozygous mutant zebrafish and the homozygous mutant zebrafish are hybridized, and homozygous double-mutant epc1- / -epc2- / -zebrafish can be obtained. The obtained epc1- / -epc2- / -environment susceptible mutant zebrafish model can be used for carrying out biological indication monitoring on the oxygen content and bacteria in a fishery water environment or a drinking water environment, is applied to real-time early warning of water environment pollution and drinking water pollution, and has the advantages of higher sensitivity, quicker emergency effect and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to epc1 - / - epc2 - / - Mutant and its application in constructing environmentally susceptible zebrafish model, the epc1 constructed by the present invention - / - epc2 - / - Environmentally susceptible mutant zebrafish can be used to establish a water environment monitoring system with the death of the organism as the monitoring endpoint. Background technique [0002] EPC1 (enhancer of polycomb homolog 1) and EPC2 (enhancer of polycombhomolog 2) are important chromatin regulators that are highly conserved from yeast to humans (Doyon et al., 2004). EPC family genes are involved in a variety of physiological processes including cell differentiation and development (Prasad et al 2014, Wang et al 2016, Searle et al 2017). In yeast, deletion of Epl1 (the yeast homolog of EPC) results in cellular accumulation at G2 / M and global loss of acetylated histones H4 and H2A, and Epl1 mutations supp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12A01K67/027
CPCC12N15/85C07K14/461A01K67/0276A01K2217/15A01K2227/40A01K2267/02A01K2267/03A01K2267/0393
Inventor 刘静霞刘茜刘文叶林舒慧
Owner HUAZHONG AGRI UNIV
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