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A method for constructing a zebrafish heart-specific expression model and related vectors

A construction method and zebrafish technology, applied in the field of genetic engineering, can solve problems such as no reports on the construction of a zebrafish heart model, and achieve the effects of easy in vivo tracking and monitoring, obvious gene expression effect, and accurate targeting.

Active Publication Date: 2022-03-29
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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  • A method for constructing a zebrafish heart-specific expression model and related vectors
  • A method for constructing a zebrafish heart-specific expression model and related vectors
  • A method for constructing a zebrafish heart-specific expression model and related vectors

Examples

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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 method for constructing a zebrafish heart-specific expression model and related vectors. The homologous recombination vector includes an insert fragment inserted into the original vector. The insert fragment includes a zebrafish heart-specific expression promoter, a left homology arm and a right homology arm fused with the LoxP site sequence, a multiple cloning site, and internal ribose body entry site, reporter gene, and transcription termination signal fragment; wherein, the promoter is located upstream of the reporter gene, the termination signal is located downstream of the reporter gene, the left homology arm is located upstream of the promoter, and the right homology arm is located at the termination Downstream of the signal, the inner side of the homology arm contains the LoxP sequence, and the multiple cloning site is located between the promoter and the reporter gene; the left homology arm and the right homology arm target the zebrafish pigment gene. A method for constructing a zebrafish heart-specific expression model is also provided. The present invention can be used to quickly establish a transgenic model of zebrafish heart-specific expression genes, and at the same time, can regulate the expression of the inserted 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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Patent Type & Authority Patents(China)
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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