Quick identification method and application of transgenic fish homozygote

An identification method and a transgenic technology, applied in the field of aquatic animal genetics and breeding, can solve the problems of time-consuming and laborious, low stability of identification results, and achieve the effects of speeding up the breeding process, excellent breeding traits, and stable genetic traits.

Inactive Publication Date: 2012-07-04
INST OF AQUATIC LIFE ACAD SINICA
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  • Abstract
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  • Claims
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Problems solved by technology

However, these methods for identifying homozygous transgenic fish are not only time-consuming and laborious, but also the stability of the identified results is not high

Method used

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  • Quick identification method and application of transgenic fish homozygote
  • Quick identification method and application of transgenic fish homozygote
  • Quick identification method and application of transgenic fish homozygote

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for identifying homozygous transgenic fish, the steps of which are:

[0031] 1. DNA extraction

[0032] Cut about 0.5-1cm 2 Genomic DNA of transgenic fish was extracted from caudal fin tissue of GH transgenic fish according to the operation guide of "Blood / Cell / Tissue Genomic DNA Extraction Kit" (product of Beijing Tiangen Biotechnology Company).

[0033] 2. Positive identification of transgenic fish

[0034] Positive identification of GH transgenic fish was carried out by conventional PCR technique. The total volume of the PCR system is 25ul, including 20ng DNA, 100uMdNTP, 2.5ulBuffer (25mM), 2.5ul MgCl 2 (25 mM), 1 Unit T. Aquaticus DNA polymerase (Fermentas, USA), and 1 μl each of primers (10 mM) (Invitrogen, the same below). Primer sequences: GHf (5'-CATTT ACAGTTCAGCCATGGCTAGA-3') and GHr (5'-AGCACCACCGACAACAGCACTA ATG-3'). Reaction program: pre-denaturation at 94°C for 5 minutes, amplification for 30 cycles (94°C, 30s; 58°C, 30s; 72°C, 30s), and final...

Embodiment 2

[0042] Application of a screened homozygous transgenic Yellow River carp in cultivating excellent cultured fish species. The specific process is:

[0043] A, the identified male transgenic Yellow River carp homozygote and the female transgenic Yellow River carp homozygote are cultured in ponds according to conventional methods, and after they develop to sexual maturity, artificial induction of labor is carried out according to conventional methods;

[0044] B. artificial insemination of the homozygous semen of the male transgenic Yellow River carp and the eggs of the female transgenic Yellow River carp;

[0045] C. The progeny obtained by self-crossing is a homozygous family of transgenic fish with stable genetic properties, and the obtained homozygous family of transgenic Yellow River carp is bred and cultivated in ponds according to conventional methods, thereby cultivating new varieties of excellent cultured fish.

[0046]

[0047]

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Abstract

The invention discloses a quick identification method and application of a transgenic fish homozygote. The method comprises the following steps: A. cutting about 0.5-1cm<2> of transgenic fish tail fin tissues, and extracting DNA (deoxyribonucleic acid); B. detecting that the transplanted gene is positive by conventional PCR technology; and C. analyzing the genotype of the transgenic fish of which the transplanted gene is positive by an SYBRGreenreal-timePCR 2<DELTADELTACt> method, comparing the integrated relative number of copies of the transplanted gene in the homozygote transgenic fish and hemizygote transgenic fish, identifying and screening out the transgenic homozygote, and further verifying by a test-mating experiment. The method is easy to implement and simple to operate, and is quick and accurate. The identification and screening of the transgenic fish homozygote have important meanings for establishing the transgenic fish pure line and accelerating the cultivation progress of new species of transgenic fish.

Description

technical field [0001] The present invention relates to the technical field of aquatic animal genetics and breeding, more specifically to a method for identifying homozygous transgenic fish for growth hormone (GH, hereinafter the same) and also relates to the establishment of homozygous transgenic fish in the pure line of transgenic fish and new varieties applications in cultivation. Using a combination of conventional PCR and quantitative PCR (real-time PCR, the same below), the genotype of the transgenic fish positive for the transgenic gene was analyzed by comparing the relative copy number of the transgenic gene integration in the homozygous and hemizygous transgenic fish , so as to quickly and accurately identify transgenic fish homozygotes, which is of great significance for establishing pure lines of transgenic fish and speeding up the process of breeding new varieties of transgenic fish. Background technique [0002] Since the birth of the first batch of fast-growin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68A01K67/027
Inventor 胡炜朱作言钟成容宋焱龙汪亚平
Owner INST OF AQUATIC LIFE ACAD SINICA
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