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Method for breeding Macropodus opercularis with transferred green fluorescent protein gene by using piggyBac transposons

A technology of green fluorescent protein and fork-tailed betta fish, which is applied in the introduction of foreign genetic material using vectors, recombinant DNA technology, animal husbandry, etc., to achieve the effect of broadening the research space

Inactive Publication Date: 2011-02-23
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] The piggyBac transposon is a powerful transgenic carrier. PiggyBac transposon-mediated transgenic research technology has been successfully reported in insects and mammals, but there is no relevant report on aquatic animals.

Method used

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  • Method for breeding Macropodus opercularis with transferred green fluorescent protein gene by using piggyBac transposons
  • Method for breeding Macropodus opercularis with transferred green fluorescent protein gene by using piggyBac transposons
  • Method for breeding Macropodus opercularis with transferred green fluorescent protein gene by using piggyBac transposons

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Experimental program
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Effect test

Embodiment 1

[0018] Using the microinjection method, the piggyBac plasmid carrying the green fluorescent protein (EGFP) marker gene (such as figure 1 shown) and their helper plasmids capable of providing transposases (such as figure 2 shown) were mixed at a ratio of 10:1, the total concentration of the two plasmids was 30 ng / μl, the plasmids were dissolved in 0.1 mM phosphate buffer containing 1 mM sodium chloride, and then within 1 hour of spawning of the fork-tailed betta fish, the The microinjection method is used to introduce into the fertilized egg, the introduction site is the animal pole of the fertilized egg, and the total volume of introduction is 5nl. G from transgenic experiments 1 Transgenic betta fish stably expressing the EGFP marker gene were screened by fluorescence microscopy (Olympus, SZX12, Japan) from the embryonic development period of the generation of Betta fish. The excitation wavelength was 460nm-490nm, and the emission wavelength was 510nm-550nm.

[0019] A tot...

Embodiment 2

[0021] Using the microinjection method, the piggyBac plasmid carrying the EGFP marker gene and its helper plasmid capable of providing transposase were mixed at a ratio of 1:2. The total concentration of the two plasmids was 900ng / μl. The plasmid was dissolved in 2mM containing 8mM Cl Sodium chloride phosphate buffer solution, and then introduced into the fertilized eggs within 1 hour of spawning of the forktail betta, and the total volume of introduction was 0.8nl. From transgenic experiment G 1 Transgenic betta fish expressing the EGFP marker gene were screened by a fluorescence microscope (Olympus, SZX12, Japan) from the embryonic development period of the generation of Betta fish. The excitation wavelength was 460nm-490nm, and the emission wavelength was 510nm-550nm.

[0022] A total of 908 fertilized eggs were injected in the transgenic experiment, and 81 betta fish were obtained, G 1 Five fork-tailed bettas in the generation were transgenic fish. In the detected G 1 I...

Embodiment 3

[0024] Using the microinjection method, the piggyBac plasmid carrying the GFP marker gene ( figure 1 ) and its transposase-providing helper plasmid ( figure 2 ) were mixed at a ratio of 3:1, the total concentration of the two plasmids was 120ng / μl, the plasmids were dissolved in 0.5mM phosphate buffer containing 3mM sodium chloride, and then microinjected within 1 hour of forktail betta spawning In the fertilized egg, the introduction site is the animal pole of the fertilized egg, and the total volume of introduction is 2nl. G from transgenic experiments 1 From the embryonic development period of the fork-tailed betta, transgenic bettas expressing the EGFP marker gene were screened by a fluorescence microscope (Olympus, SZX12, Japan). The excitation wavelength was 460nm-490nm, and the emission wavelength was 510nm-550nm.

[0025] A total of 873 fertilized eggs were injected, and 119 fork-tailed betta fish were obtained, G 1 Six fork-tailed bettas in the generation were tra...

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Abstract

The invention discloses a method for breeding Macropodus opercularis with a transferred green fluorescent protein gene by using piggyBac transposons, which comprises the following steps of: firstly, constructing piggyBac transposons with green fluorescent protein genes and a helper plasmid capable of providing transposase; and secondly, transferring the two transposons into a germ cell of the Macropodus opercularis by using microinjection technology, and according to the transposons characteristics of the piggyBac transposons, introducing the green fluorescent protein genes into a genome of the Macropodus opercularis so as to make the gene inherited and expressed stably. An individual of transgenic Macropodus opercularis can be screened by means of the fluorescence-labeled gene, and the Macropodus opercularis with the transferred green fluorescent protein gene is more worthy of appreciation and represents a new variety of Macropodus opercularis with specific function. In addition, it is the first time for the piggyBac transposons to be used in researches on transgenic fish, and thus, the research space for transgenic aquatic animals is widened.

Description

technical field [0001] The invention relates to a method for creating a green fluorescent protein gene-transferred forktail betta by using a piggyBac transposon. Background technique [0002] The piggyBac transposon is a powerful transgenic carrier. PiggyBac transposon-mediated transgenic research technology has been successfully reported on insects and mammals, but there is no relevant report on aquatic animals. The fork-tailed betta Macropodus opercularis (Linnaeus) belongs to the Perciformes family Anabantidae. It is a small inland freshwater ornamental fish widely distributed in southern my country and Southeast Asian countries. It has the advantages of wide source, easy generation and easy breeding. The invention introduces the piggyBac transposon into the transgenic technology of aquatic animals, uses the piggyBac transposon as a carrier, and integrates the green fluorescent protein gene into the genome of the fork-tailed betta. This technology can not only improve the...

Claims

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Application Information

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IPC IPC(8): C12N15/63A01K67/027C12N15/65
Inventor 杨国梁高强危浩庄兰芳王军毅叶少群钟伯雄
Owner ZHEJIANG UNIV
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