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A method for developing an ecologically safe transgenic fluorescent ornamental fish

An ornamental fish and transgenic technology, applied in the fields of genetic engineering and molecular breeding of ornamental fish, can solve ecological risks and other problems

Active Publication Date: 2019-03-01
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the presence of genetically modified components in the gonads of the current fluorescent ornamental fish, the offspring of ornamental fish can breed and pass on the genetically modified elements, so there are certain ecological risks

Method used

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  • A method for developing an ecologically safe transgenic fluorescent ornamental fish
  • A method for developing an ecologically safe transgenic fluorescent ornamental fish
  • A method for developing an ecologically safe transgenic fluorescent ornamental fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of blue fluorescent protein expression vector pISceI-LoxP-CMV-eCFP

[0048] Such as figure 1 As shown, the zebrafish blue fluorescent protein expression vector pISceI-LoxP-CMV-eCFP with LoxP site was constructed by PCR method.

[0049] Step S1, preparation of insert fragments

[0050] Using the vector pISceI-CMV-eCFP as a template, the CMV-R-SV40 fragment was amplified by PCR, and LoxP sites were introduced at both ends of it. The upstream and downstream primers for PCR amplification are respectively LoxP-KpnI-F (shown in SEQ ID NO.5) and LoxP-SacI-R (shown in SEQ ID NO.6).

[0051] The required PCR reaction program is: 94°C for 5min; 30 cycles of 94°C for 20s, 55°C for 30s, and 72°C for 2min; 72°C for 5min. After the PCR product was detected by 1% agarose gel electrophoresis, the product was purified with a PCR product purification kit to remove impurities such as salt ions, enzymes and primers in the PCR reaction system. The purified produc...

Embodiment 2

[0058] Example 2: Construction of the green fluorescent protein expression vector pISceI-LoxP-CMV-eGFP

[0059] Such as figure 2 As shown, the green fluorescent protein expression vector pISceI-LoxP-CMV-eGFP with LoxP site was constructed by PCR method. Its similarity with Embodiment 1 will not be repeated.

[0060] Step S1, preparation of insert fragments:

[0061] The laboratory's existing vector pISceI-CMV-eGFP was used as the template for PCR amplification, and the rest of the operations were the same as in Example 1. Inserts are recovered.

[0062] Step S2, preparing carrier skeleton

[0063] Plasmid pISceI-CMV-eGFP was digested with KpnI and SacI at the same time, and other operations were the same as in Example 1. Recover the carrier skeleton.

[0064] Step S3, preparation of vector pISceI-LoxP-CMV-eGFP

[0065] Operation is the same as embodiment one. The ligated product was verified to be the green fluorescent protein expression vector pISceI-LoxP-CMV-eGFP. ...

Embodiment 3

[0066] Example 3: Construction of red fluorescent protein expression vector pISceI-LoxP-CMV-mCherry

[0067] Such as image 3 As shown, the red fluorescent protein expression vector pISceI-LoxP-CMV-mCherry with LoxP site was constructed by PCR method. Its similarity with Embodiment 1 will not be repeated.

[0068] Step S1, preparation of insert fragments:

[0069] The laboratory's existing vector pISceI-CMV-mCherry was used as the template for PCR amplification, and the rest of the operations were the same as in Example 1. Inserts are recovered.

[0070] Step S2, preparing carrier skeleton

[0071] Plasmid pISceI-CMV-mCherry was digested with KpnI and SacI at the same time, and other operations were the same as in Example 1. Recover the carrier skeleton.

[0072] Step S3, preparation of vector pISceI-LoxP-CMV-mCherry

[0073] Operation is the same as embodiment one. The ligation product was confirmed to be the red fluorescent protein expression vector pISceI-LoxP-CMV-m...

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Abstract

The invention discloses a fluorescent protein expression vector, methods for constructing and applying the fluorescent protein expression vector, a sexual gonad tissue specific regulation and expression vector, methods for constructing and applying the sexual gonad tissue specific regulation and expression vector and a method for cultivating ecological safety type transgenic fluorescent ornamental fish. Fluorescent proteins can be driven by the aid of promoters CMV with whole-body general expression functions to be expressed in the ornamental fish, and whole-body expression functions of the ornamental fish can be realized by the aid of the fluorescent protein expression vector. Reverse tetracycline-controlled transactivators rtTA can be driven by sexual gonad specific promoters kop to be expressed, and TRE promoters can be driven to start downstream Cre protein expression plasmid vectors after the reverse tetracycline-controlled transactivators rtTA and tetracycline are combined with one another. Sexual gonad of the ecological safety type transgenic fluorescent ornamental fish cultivated by the aid of the method does not contain transgenic components after the ecological safety type transgenic fluorescent ornamental fish is authenticated.

Description

technical field [0001] The invention relates to gene recombination vector, construction method and application. In particular, a fluorescent protein expression vector and a gonad tissue-specific regulatory expression vector, as well as the application of the vector in cultivating ecologically safe transgenic fluorescent ornamental fish. The invention belongs to the fields of genetic engineering and ornamental fish molecular breeding. Background technique [0002] Nowadays, the cultivation of fluorescent ornamental fish has formed a huge industrial market on a global scale. The fluorescent ornamental fish is different from the traditional ornamental fish. It is an artificially cultivated ornamental fish species that emits different fluorescence through the fluorescent protein carried by itself under the condition of a certain wavelength of light. Fluorescent ornamental fish has higher ornamental value and experimental research value. For example, the zebrafish strain expre...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
Inventor 裴得胜边万平
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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