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Construction method and related vector of zebra fish heart specific expression model

A zebrafish-specific technology, applied in the field of genetic engineering, can solve problems that have not yet been reported on the construction of a zebrafish heart model

Active Publication Date: 2018-12-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report on the construction of zebrafish heart model

Method used

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  • Construction method and related vector of zebra fish heart specific expression model
  • Construction method and related vector of zebra fish heart specific expression model
  • Construction method and related vector of zebra fish heart specific expression model

Examples

Experimental program
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Effect test

Embodiment 1

[0042] This Example 1 provides a protocol for expressing the EGFP gene in the heart through a heart-specific promoter. The transgene homologous recombination vector pZF-LoxP-HMG-PA containing the EGFP reporter gene was constructed, and the double-strand gap mediated by CRISPR / Cas9 was used to insert it into the first exon region of the Tyrosinase gene by the MMEJ method. The expression of the gene was inserted into the reporter gene at the same time, and finally through the double screening of the reporter gene and the pigment gene, the zebrafish individuals whose heart expressed the target gene were obtained.

[0043] Present embodiment 1 specifically comprises the following steps:

[0044] 1. Construct a homologous recombination vector pZF-LoxP-HMG-PA containing a reporter gene, and obtain a fragment that can be used for zebrafish injection.

[0045] 1.1 Design of targeting sites and upstream and downstream homology arms of homologous recombination.

[0046] Our laboratory...

Embodiment 2

[0093] In order to confirm that the cloned gene can be inserted into the MSC site of the vector pZF-LoxP-HMG-PA constructed in this study, the selected gene is the zebrafish TRDMT1 gene (NCBI: NM_001018143.1), and the cloned CDs sequence is inserted into the The multiple cloning site of the vector, the insertion is identified by PCR.

[0094] (1) The vector is amplified and then digested for later use

[0095] The carrier strain was inoculated into LB liquid medium, cultured at 37°C overnight, the plasmid was extracted with OMEGA plasmid extraction kit, digested with BglII, and set aside. The digestion method is shown in the example below.

[0096] 5'-A↓G A T C T-3'

[0097] 3'-T C T A G↑A-5'

[0098] (2) Obtain the CDs sequence inserted into the TRDMT1 fragment

[0099] The CDs sequence of 1146bp TRDMT1 gene was amplified from zebrafish cDNA. The amplification primers were TR-F and TR-R, and a restriction enzyme site was added at the 5' end of the primers. The amplificati...

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Abstract

The invention discloses a construction method and a related vector of a zebra fish heart specific expression model. A homologous recombinant vector comprises an insert fragment which is inserted withan initial vector and comprises a promoter of zebra fish heart specific expression, left and right homologous arms fused with LoxP site sequences, a polyclonal site, an internal ribosome entry site, areporter gene and a transcription termination fragment, wherein the promoter is positioned at the upstream of the reporter gene, and the termination signal is positioned at the downstream of the reporter gene; the left homologous arm is positioned at the upstream of the promoter, and the right homogenous arm is positioned at the downstream of the termination signal; the inner sides of the homologous arms contain LoxP sequences; the polyclonal site is positioned between the promoter and the reporter gene; and the left and right homologous arms are targeted to the zebra fish pigment gene. The invention further provides a construction method of the zebra fish heart specific expression model, which can be used for quickly establishing a transgenic model of the zebra fish heart specific expression gene, and regulating expression of the insert fragment.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing a zebrafish heart-specific expression model and related vectors. Background technique [0002] Zebrafish is a widely used model animal and plays an important role in the field of developmental biology. Zebrafish has many advantages in the study of embryonic development. Its embryonic development is fast, the organ development process is obvious, and it is easy to track and detect. The heart model, brain model, liver model, eye model, etc. that have been used so far are popular animal models, especially in the field of developmental biology. With the application of two-photon interference microscopy, in vivo real-time observation has become an effective means. [0003] In molecular biology research, the use of CRISPR / Cas9 technology to generate double-strand deletions of DNA provides new pathways for gene knockout, gene knockin, and various genome rea...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/90A01K67/027
CPCA01K67/0278C12N15/11C12N15/63C12N15/902A01K2227/40A01K2217/075A01K2207/15A01K2267/0375
Inventor 崔恒宓薛松磊沈晖孙振欧阳娟徐慧
Owner YANGZHOU UNIV
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