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Method for creating green fluorescent protein transgenic macropodus opercularis by using pRC/CMV-EGFP plasmids

A technology of CMV-EGFP and green fluorescent protein, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, animal husbandry, etc., to achieve the effect of enhancing ornamental value and broadening the research space

Inactive Publication Date: 2011-07-13
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many research reports on human medicine and livestock and poultry research on transgenic research using pRC / CMV as a vector, and there are also research reports on aquatic animals (such as zebrafish), but there are no related reports on forktail betta

Method used

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  • Method for creating green fluorescent protein transgenic macropodus opercularis by using pRC/CMV-EGFP plasmids
  • Method for creating green fluorescent protein transgenic macropodus opercularis by using pRC/CMV-EGFP plasmids
  • Method for creating green fluorescent protein transgenic macropodus opercularis by using pRC/CMV-EGFP plasmids

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Experimental program
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Effect test

Embodiment 1

[0016] Using the microinjection method, the pRC / CMV-EGFP plasmid carrying the green fluorescent protein (EGFP) marker gene ( figure 1 ) was adjusted to a concentration of 40ng / μl, the plasmid was dissolved in 0.1mM phosphate buffer containing 8mM sodium chloride, and then introduced into the fertilized eggs by microinjection within 1 hour of laying eggs, and the introduction site was fertilized The egg animal pole was introduced into a total volume of 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.

[0017] A total of 1338 fertilized eggs were injected, G 0 Generation obtained 215 forktails, G 1 In the generation, 14 forktail betta fish were transgenic fish. In the detected G 1 EGFP was expressed in the...

Embodiment 2

[0019] Using the microinjection method, the concentration of the pRC / CMV-EGFP plasmid carrying the EGFP marker gene was 250 ng / μl, and the plasmid was dissolved in 2 mM phosphate buffer containing 1 mM sodium chloride, and then spawned in fork-tailed betta fish for 1 hour Introduced into fertilized eggs, the total volume of introduction is 0.5nl. 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.

[0020] A total of 1188 fertilized eggs were injected in the transgenic experiment, and 220 fork-tailed betta fish were obtained, G 1 In the generation, 13 forktail betta fish were transgenic fish. In the detected G 1 In the transgenic forktail betta individuals, there were green fluorescent expressions in the tail (2 tails),...

Embodiment 3

[0023] The pRC / CMV-EGFP plasmid carrying the EGFP marker gene ( figure 1 ) at a concentration of 150ng / μl, the plasmid was dissolved in 0.6mM phosphate buffer containing 4mM sodium chloride, and microinjected into fertilized eggs within 1 hour of laying eggs in Forktail Betta, the introduction site was the animal pole of fertilized eggs, The total volume introduced was 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.

[0024] A total of 971 fertilized eggs were injected, and 219 fork-tailed betta fish were obtained, G 1 In the generation, 14 forktail betta fish were transgenic fish. In the detected G 1 In the transgenic forktail betta individuals, EGFP was expressed in the tail (2 tails), eyes (11 tails) and intestine (...

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Abstract

The invention discloses a method for creating green fluorescent protein transgenic macropodus opercularis by using pRC / CMV-EGFP plasmids. The method comprises the following steps of: constructing the pRC / CMV-EGFP plasmids with green fluorescent protein genes, guiding the plasmids into fertilized eggs of macropodus opercularis by using a microinjection technology, namely guiding the green fluorescent protein genes into genomes of the macropodus opercularis, and performing stable inheritance and expression to obtain the transgenic macropodus opercularis. The transgenic macropodus opercularis can be used for gene function research and promotes the ornamental value.

Description

technical field [0001] The invention relates to a method for creating a transgenic fork-tailed betta by utilizing the pRC / CMV-EGFP plasmid. Background technique [0002] pRC / CMV is a eukaryotic expression vector with a strong CMV promoter, and also a powerful transgenic vector with a CMV promoter, a transcription termination signal derived from BGH, and an SV40 origin of replication. Transgenic studies using pRC / CMV as vectors have been widely reported in human medicine, livestock and poultry, and also in aquatic animals (such as zebrafish), but there are no related reports on forktail betta. 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 present invention introduces the pRC / CMV eukaryotic expression vector and EGFP into the...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65A01K67/027C12Q1/68
Inventor 钟伯雄高强杨国梁龙勇危浩王军毅庄兰芳叶少群
Owner ZHEJIANG UNIV
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