Zebrafish Nerve Tissue-Specific Enhancer and Its Cloning and Application

A neural tissue, specific technology, applied in the field of enhancer capture, can solve the problem of few specific enhancers

Active Publication Date: 2017-12-29
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the problem of fewer nerve tissue-specific enhancers in the existing research, and provide a zebrafish nerve tissue-specific enhancer and its cloning and application, that is, to establish nervous system-specific expression through enhancer capture vectors Fluorescent transgenic zebrafish cloned with an enhancer sequence specifically expressed in neural tissue

Method used

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  • Zebrafish Nerve Tissue-Specific Enhancer and Its Cloning and Application

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Experimental program
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Effect test

Embodiment 1

[0038] Example 1, construction of enhancer capture vector:

[0039] ① Design primers to amplify the mMT1 minimal promoter element from the pmMREd12mt1LUC3 plasmid, the required primer sequences:

[0040] Upstream primer: 5'-ATATTCGTACGACTCGTCCAACGACTATAA-3'(NO.3);

[0041] Downstream primer: 5'-CGGGGTACCGGTGAAGCTGGAGCTACG-3' (NO.4).

[0042] ②Using the pmMREd12mt1LUC3 plasmid as a template and using the above primers, a 105bp fragment was amplified. PCR cycle conditions: 94°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 30s, 30 cycles; 72°C for 10min, PCR amplified product It was recovered by 1% agarose gel electrophoresis kit.

[0043] ③ After the above PCR product was recovered and purified by gel, it was digested with restriction endonucleases BsiWI and KpnI, ligated with the pEF1ɑ-EGFP vector fragment that had undergone the same digestion, and transformed into Escherichia coli Top10'competent cells, and treated with ampicillin ( Amp)-LB plate screening to obtain posit...

Embodiment 2

[0049] Embodiment 2, zebrafish rearing and transgene microinjection

[0050] ① Breeding of zebrafish: zebrafish (Danio rerio), strain AB, feeding condition 28°C, 12h light / 12h night.

[0051] ② Synthesis of Tol2 transposase capped mRNA

[0052] A. Linearization: Use BamHI to digest the vector used for linearized transcription;

[0053] B. Use the BioFluxDNA Gel Recovery Kit to recover the linearized template, and dissolve the linearized template in DEPC water;

[0054] C. Transcription: T7 transcriptase (Ambion, Foster City, CA) in the mature mRNA synthesis kit was used for transcription. The transcription system is as follows:

[0055]

[0056] After mixing the above components, transcribe at 37°C for 2 hours;

[0057] D. After taking 1 μL for electrophoresis detection, add 0.5 μL 2U / μL DnaseI, and incubate at 37°C for 15 minutes;

[0058] E. Add 57.5 μL Nuclear-free water and 7.5 μL NH4AC to terminate the reaction;

[0059] F. Equal volume of phenol chloroform / chloro...

Embodiment 3

[0067] Example 3, Positive Screening of Transgenic Zebrafish

[0068] The sexually mature zebrafish of the F0 generation was crossed with the wild-type zebrafish, and the obtained F1 generation was observed with green fluorescence using a Carl Zeiss SteReo LumarV12 stereoscopic fluorescence microscope; the excitation wavelength of green fluorescent protein was 488nm, and the emission wavelength was 507nm, using Zeiss fluorescence inverted microscope was used to observe the fluorescence of F1 generation embryos at 12hpf, 24hpf, 48hpf, 72hpf, 4day, and 5day stages to obtain zebrafish embryos with specific green fluorescent protein expression in the nervous system; the fluorescent embryos were picked out and raised until Adult zebrafish are further crossed with wild-type zebrafish to obtain sufficient transgenic zebrafish heterozygous F2; ​​heterozygous self-crossing can obtain fluorescently labeled homozygous F3.

[0069] Laser confocal microscopic observation and whole-mount in...

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Abstract

The invention discloses a zebrafish nerve tissue-specific enhancer and its cloning and application, and relates to the technical field of enhancer capture of tissue- and organ-specifically expressed genes, which is used for fluorescently labeling nerve cells in the development of zebrafish embryos . The zebrafish nerve tissue-specific enhancer has a sequence of the nucleotide sequence shown in SEQ ID NO.2. Its application is: ① develop transgenic fish with specific expression of fluorescence in nervous tissue, which can be used for research on the regeneration process of nervous organs; ② used to drive the specific expression of any target gene in nervous tissue, providing a powerful tool for researchers to study nervous system development and related disease treatment . The invention can be used in the research of zebrafish eye development and damage regeneration process; it is helpful to study the function of specific gene in nervous system development and regulation, and provides new materials for the treatment of related diseases.

Description

technical field [0001] The present invention relates to the technical field of enhancer capture of tissue- and organ-specifically expressed genes, in particular to a zebrafish nerve tissue-specific enhancer and its cloning and application, which are used to fluorescently label nerve cells in the development of zebrafish embryos . Background technique [0002] Enhancer capture technology is one of the forward genetics methods to study the enhancer sequences existing in the genome, and it is used to discover known and unknown tissue-specific enhancer elements. Over the past few decades, enhancer capture technology has been used at home and abroad to obtain zebrafish strains that specifically express fluorescence in various tissues and organs, but little information has been obtained on the insertion of related gene sites. Many tissues and organs such as the nervous system, liver, and pancreas and kidney-specific enhancers and promoters have not yet been cloned. Nervous syste...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85A01K67/027
Inventor 宋桂丽刘春艳龙勇李青崔宗斌
Owner INST OF AQUATIC LIFE ACAD SINICA
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