Fish gene transfer vector based on Tgf2 transposons and preparation method thereof

A gene transfer and transposon technology, applied in the field of fish genetic engineering, can solve problems such as inactivation of transposase, and achieve the effects of high cost, high integration and low cost

Inactive Publication Date: 2011-02-02
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are also many transposons in vertebrates, but the transposases of such transposons are mostly inactive during the long evolution

Method used

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  • Fish gene transfer vector based on Tgf2 transposons and preparation method thereof
  • Fish gene transfer vector based on Tgf2 transposons and preparation method thereof
  • Fish gene transfer vector based on Tgf2 transposons and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, Construction of Transgene Donor Plasmid pTgf2-zfKrt8-eGFP

[0033]Zebrafish (Danio rerio, C202, School of Fisheries and Life Sciences, Shanghai Ocean University) was clipped to extract genomic DNA, and stored at -20°C for future use. Design primers SEQ ID No: 5 (with BamHI restriction site) and SEQ ID No: 6 (with XhoI restriction site) for PCR amplification in the zebrafish genome, the amplification reaction program is: 94 ° C for 5 min; 94 ° C 30s, 55°C for 30s, 72°C for 150s, 35 cycles; 72°C for 5min. The amplified products were separated by electrophoresis at a voltage of 5V / cm on 1.2% agarose gel with TBE as a buffer, and the target fragments were recovered by tapping the gel, and the recovered products were connected to pMD-19T vector (TaKaRa) respectively, and transfected Enter Escherichia coli DH5α, pick positive clones, inoculate into liquid LB medium with ampicillin content of 50 μg / mL, culture overnight at 37°C with shaking, take 200 μL of bacteria...

Embodiment 2

[0035] The construction of embodiment 2, Tgf2 transposase (gfTP) helper plasmid

[0036] According to the information obtained, the transposase (gfTP) encoded by the Tgf2 transposon has the highest expression level in the mature ovary of goldfish (Ryukin goldfish Carassius auratus var.). Total RNA was extracted, reversed into 3'-end single-stranded cDNA with reverse transcriptase in TaKaRa RNA PCR Kit (AMV) Ver.3.0, and stored at -20°C for future use. With forward and reverse primers SEQ ID No: 1 (with XhoI restriction site) and SEQ ID No: 2 (with XbaI restriction site) PCR amplified Tgf2 transposase coding region 1734bp full length, the amplified product after 1.2% Agarose gel, using TBE as a buffer, after electrophoresis separation at a voltage of 5V / cm, the target fragment was recovered by tapping the gel, and the recovered product was connected to the pMD-19T vector (TaKaRa company), and transformed into Escherichia coli DH5α, picked Positive clones were taken and inocula...

Embodiment 3

[0040] Example 3, Transgenic effect of Tgf2 transgenic elements and helper plasmids in zebrafish

[0041] Breeding of zebrafish: The zebrafish used in the experiment were raised in a recirculating aquarium, and the male and female were kept separately. They were fed twice a day, once with Artemia, and once with artificial feed. The culture temperature was about 28°C, with 14 hours of light and 10 hours of darkness. . The night before spawning, the female fish and the male fish are transferred into the spawning tank, and the female and the male fish are separated by a transparent baffle. On the morning of the experiment, the baffle was removed to allow it to lay eggs naturally.

[0042] Microinjection: take pTgf2-zfKrt8-eGFP (see figure 2 ) transgene donor plasmid and the Tgf2 transposase helper plasmid pCS2-zfβ-actin-gfTP encoded by the Tgf2 transposon (see Figure 4 ), using 0.3×Danieau buffer (containing 0.1% phenol red) to prepare mixed solutions with a final concentrat...

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Abstract

The invention discloses a simple, convenient and high-efficiency transgene component based on fish Tgf2 transposons and a preparation method thereof. The transgene component of the invention is formed by a transgene donor plasmid and an auxiliary plasmid, wherein the transgene donor plasmid is established by a fish promoter sequence, such as a zebra fish Krt8 promoter, target genes, and left and right arm sequences of goldfish Tgf2 transposons; and the auxiliary plasmid is established by a zebra fish belta-actin promoter sequence and coding genes of goldfish Tgf2 transposons enzymes. Compared with the prior art, the invention has the characteristics of simple operation, high conversion efficiency and low cost and provides a new method for fish transgene research.

Description

technical field [0001] The invention relates to constructing a gene transfer vector based on fish Tgf2 transposon, and also discloses the construction method of the transgene donor plasmid and transposase auxiliary plasmid vector involved in the vector, and its application in zebrafish and cultured fish in the application. The invention belongs to the field of fish genetic engineering. Background technique [0002] With the improvement of people's living standards, people have a greater demand for high-quality aquatic products including fish, shrimp, shellfish and algae. It is an extremely effective way to improve the growth, stress resistance and meat quality of aquaculture objects by transgenic germplasm. As early as 1984, my country's Zhu Zuoyan and others developed the world's first batch of transgenic fish and established a theoretical model of transgenic fish. Despite the remarkable achievements in aquatic transgenic research, there are still many shortcomings. For ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/85
Inventor 邹曙明蒋霞云杜雪地沈睿杰袁剑
Owner SHANGHAI OCEAN UNIV
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