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Medaka ovary structural protein gene promoter and applications thereof

A technology of ovarian structural proteins and promoters, applied in the direction of microorganism-based methods, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc., can solve the situation of time-consuming, labor-intensive, expensive, and impossibility of gonadal hermaphroditism And other issues

Active Publication Date: 2014-02-19
PEKING UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

[0003] At present, the evaluation of the occurrence of hermaphroditism in fish mainly relies on the method of tissue section observation, but this method is time-consuming and labor-intensive, requires skilled technology, is very expensive, and often only a few sides of the gonads can be observed, which cannot be done. Accurate and comprehensive evaluation of the hermaphroditism of the whole gonad (Lin et al. Ecotox Environ Safe 72:286-292.)
This greatly limits our understanding of hermaphroditism in fish
At present, there is no sensitive, simple and accurate experimental method to monitor the hermaphroditism of fish in the world

Method used

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  • Medaka ovary structural protein gene promoter and applications thereof
  • Medaka ovary structural protein gene promoter and applications thereof
  • Medaka ovary structural protein gene promoter and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0015] 1. Genomic DNA Extraction

[0016] A. Cut the gonads of mature female medaka fish and grind them in 1ml Trizol reagent until the solution is homogenized.

[0017] B. Add 200 μl of chloroform to the homogenate, mix well, and centrifuge at 12000 rpm, 4° C., for 15 min.

[0018] C. After removing the centrifuged supernatant, add 400 μl of DNA extraction buffer to the centrifuge tube, and incubate at 55° C. for 10 minutes. Mix well, centrifuge at 12000rpm, 4°C, 15min.

[0019] D. Take the supernatant, add 1ml of absolute ethanol, mix well, centrifuge at 12000rpm, 4°C, 10min.

[0020] E. Remove the supernatant, add 1ml 75% DEPC alcohol to wash the precipitate, centrifuge at 7500rpm, 4°C, 5min.

[0021] F. Remove the supernatant, dry the precipitate slightly, dissolve it in 200ml DEPC water with pH ≈ 8.0, and obtain genomic DNA.

[0022] 2. Cloning of OSP1 gene promoter

[0023] A. According to http: / / www.ensembl.org / search the DNA coding sequence of the upstream part o...

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Abstract

The present invention discloses a medaka ovary structural protein (OSP1) gene promoter and applications thereof. According to the present invention, the promoter is linked to an upstream region of green fluorescent protein gene in PEGFP-1 plasmid, and a microinjection method is adopted to inject the fusion plasmid into the fertilized egg of the medaka to construct to obtain the transgenic medaka, wherein incidence rate and occurrence degree of medaka hermaphrodite caused by exposure under exogenous endocrine interference substances can be accurately, rapidly and sensitively indicated, and the promoter can be applicable for rapid estrogen substance screening and estrogen effect substance detection in an environment.

Description

technical field [0001] The present invention relates to animal genetic engineering technology, in particular to the cloning of the promoter sequence of the medaka fish ovarian structural protein (OSP1) gene and the transgenic medaka fish established on the basis of specifically indicating the exposure of estrogen substances. Application of hermaphroditism in fish. Background technique [0002] Environmental estrogens such as some natural and synthetic estrogens, some pesticides, herbicides, etc. have been proven to cause fish gonadal retardation, testicular degeneration and hermaphroditism at low concentrations (Gimeno et al. Aquat Toxicol 43:93-109.; Papoulias et al. Environ Health Perspect 111:29-32.; Kidd et al. Proc Natl Acad Sci U.S.A. 104:8897-8901.). Fish hermaphroditism, in particular, can be observed in a variety of freshwater and marine ecosystems and has become a clear indicator of feminization in wild fish (Jobling et al. EnvironSci Technol 32: 2498-2506.; Hinck...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N15/85A01K67/027G01N33/68C12R1/19
Inventor 胡建英赵砚彬夏爽蒋洁琼
Owner PEKING UNIV
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