Fish transgenosis breeding method

A technology of transgenic and fish, applied to other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve the problems of reports without in-depth research on the specific process, and achieve the effect of easy retention

Inactive Publication Date: 2010-01-27
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Because most of the exogenous DNA fragments enter the genome and are eventually degraded, but the specific process has not been reported in depth

Method used

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  • Fish transgenosis breeding method
  • Fish transgenosis breeding method

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Experimental program
Comparison scheme
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Embodiment Construction

[0025] 1. Genomic DNA extraction and DNA rearrangement fragment construction:

[0026] Genomic DNA is extracted from carp (muscle or blood) (usually proteinase K method).

[0027] The extracted DNA was first digested with a restriction endonuclease Msp I (reaction system: 1-10 μg template DNA, 2U enzyme, 15 μL 10×Tango buffer in a total reaction volume of 150 μL; digestion at 37°C for 16 h), after the reaction was completed Precipitate with 2 times the volume of ethanol, centrifuge at 12,000 rpm, dry the ethanol, and reconnect with 30 μL volume (reaction system: 2.5U T4 ligase, 2 μL 10×linkagese buffer; connect overnight at 16°C), after the reaction is completed, use 2 Double the volume of ethanol precipitation, centrifuge at 12000 rpm, dry the ethanol, and then digest with the second restriction endonuclease EcoR I (reaction system: 50 μL total reaction volume containing 4U enzyme, 5 μL 10×Tango buffer; 37 Digested at ℃ for 16 h), precipitated with 2 times the volume of etha...

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Abstract

The invention relates to a fish transgenosis breeding method which belongs to the technical field of molecule biology fish breeding. The method mainly comprises the following steps: preprocessing the genome DNA of a fish and obtaining a DNA rearrangement fragment distinguished from the original genome DNA; guiding the DNA rearrangement fragment into an oocyte of an original fish by a transgenosis method to achieve the function with the genome of the original fish; forming a variable gene and generating individuals with mutational appearance; screening the individual with favorable production characteristics and carrying out further breeding. The invention judges whether the DNA rearrangement fragment enters the genome of the original fish or not by a TRAP method, obtains the variable gene by a two-step amplification method, provides a base for carrying out functional genome research later and obtains a unique molecular mark. The method is simple and can be widely applied to the breeding of fishes, other animals and plants, and the like and the research of relevant functional genomes.

Description

technical field [0001] The invention discloses a fish transgenic breeding method, which belongs to the technical field of molecular biology fish breeding. In the present invention, the genome DNA of a certain fish itself is pretreated to obtain DNA rearrangement fragments different from the original genome DNA, and the DNA rearrangement fragments are introduced into the egg cells of the original species of fish by transgenic methods, and the original species The genome of fish works to form mutated genes and individuals with mutations in appearance, from which excellent individuals can be screened for further breeding. The target region amplified polymorphism (TRAP) method was used to judge whether the DNA rearrangement fragment entered the genome, and the mutated gene was obtained by the two-step amplification method. Provide a basis for future functional genomics research while obtaining unique molecular markers. Background technique [0002] The technology of introducin...

Claims

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Application Information

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IPC IPC(8): C12N15/87
Inventor 曹哲明丁炜东杨健
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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