Preparation method of transgenetic zebrafish used for detecting environment pollutants
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A technology for detecting the environment and zebrafish, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, animal husbandry, etc., can solve the problem of lack of broad spectrum
Active Publication Date: 2014-03-26
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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Compared with traditional environmental detection methods, the transgenic zebrafish model has the advantages of quickness, convenience, cost-effectiveness, etc. However, the existing monitoring models often only respond to specific chemical pollutants and lack broad-spectrum
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Embodiment 1
[0019] Embodiment 1-Preparation method of transgenic zebrafish
[0020] (1) Clone the promoter regulatory sequence of the Mdr1 protein from the zebrafish genome, i.e. a DNA sequence of 280 bp upstream of the 5' end of the ATG start codon of the Mdr1 protein-encoding gene;
[0021] Through the comparison and analysis of the BLASTIN program between the mRNA sequence and the zebrafish genome sequence, the promoter sequence of the zebrafish Mdr1 gene was found on GENEBANK, and the corresponding forward and reverse primers were designed. A fragment was amplified by high-fidelity PCR, and this fragment was further ligated into the pGME-T vector. After sequencing, it was confirmed that the clone was the target fragment.
[0022] (2) Construction of expression vector mdr1promoter-GFP
[0023] The zebrafish Mdr1 promoter 280bp was excised from the vector with XhoI and BamHI, and loaded into the pEGFP vector. The resulting recombinant expression vector mdr1promoter-GFP contained the re...
Embodiment 2 1
[0028] Example 2—Verification of environmental monitoring effect of transgenic zebrafish
[0029] (1) Preparation of zebrafish culture solution containing a series of concentrations of cadmium chloride (Cd)
[0030] The zebrafish culture medium containing 10ug / L, 100ug / L, 1000ug / L Cd and the blank zebrafish culture medium without Cd were prepared respectively.
[0031] (2) Heavy metal Cd induces the expression of zebrafish fluorescent protein
[0032] After 24hpf pre-cultivation, the zebrafish embryos were placed in the culture medium containing a series of concentrations of Cd. After 5hpf, the embryos were examined under a fluorescence microscope. It could be verified that the embryos expressed stronger green fluorescence with the increase of the Cd concentration. Other heavy metals can also induce the expression of fluorescent proteins in zebrafish.
[0033] (3) PCR verification
[0034] Extract embryonic DNA pretreated by Cd, and detect the expression of GFP in it by PCR...
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Abstract
A disclosed preparation method of transgenetic zebrafish used for detecting environment pollutants comprises: (1) cloning the promoter regulatory sequence of Mdr1 protein from zebrafish genome; (2) fusing the DNA fragment obtained by cloning into the upstream of GFP coding sequence for constructing an expression vector; (3) performing zebrafish embryo microinjection on the expression vector; (4) culturing the embryo subjected to injection to sexual maturity, and enabling cultured zebrafish and wide zebrafish to copulate and oviposit; and (5) screening positive F1 generation under a fluorescence microscope, and performing inbreeding to obtain F2 generation and further to obtain a transgenic line with stable inheritance. Because the expression level of fluorescin can be induced in a concentration dependent way by multiple environment pollutants such as heavy metals, polycyclic aromatic hydrocarbons and the like, the transgenic zebrafish prepared by employing the preparation method can be used to detecting the existence and the concentration of the above chemical pollutants in environment; and the preparation method has the advantages of being economic, rapid, convenient and accurate.
Description
technical field [0001] The invention relates to a production method of an environmental pollutant biological detector, in particular to a method for constructing a transgenic zebrafish that can sensitively monitor the concentration of environmental pollutants. Background technique [0002] With the development of society, more and more synthetic chemical substances are produced and utilized by human beings. These substances enter the environment through various channels, which will not only cause the death of aquatic organisms and the destruction of aquatic ecosystems, but also directly or indirectly threaten human health. . According to estimates by the World Health Organization, chemical pollutants such as heavy metals, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls cause millions of deaths in the world every year, and are the cause of 60-90% of cancers. In China alone, the number of new cancer patients caused by industrial pollutants is as high as 945,00...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A01K67/027
Inventor 殷建林颖胡军
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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