Zebrafish nervous tissue-specific enhancer and cloning and application thereof

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

Active Publication Date: 2015-11-25
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...

Method used

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  • Zebrafish nervous tissue-specific enhancer and cloning and application thereof
  • Zebrafish nervous tissue-specific enhancer and cloning and application thereof
  • Zebrafish nervous tissue-specific enhancer and cloning and application thereof

Examples

Experimental program
Comparison scheme
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] ② Use the pmMREd12mt1LUC3 plasmid as a template and use the above primers to amplify a 105bp fragment. 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 positive col...

Embodiment 2

[0049] Embodiment 2, zebrafish rearing and transgene microinjection

[0050] ① Breeding of zebrafish: zebrafish (Daniorerio), 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, FosterCity, CA) in the mature mRNA synthesis kit is 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 / μLDnaseI, 37 ° C for 15 min;

[0058] E. Add 57.5 μL Nucleare-freewater and 7.5 μL NH4AC to terminate the reaction;

[0059] F. Extraction with equal volume of phenol chloroform / chloroform, ...

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 by using a CarlZeissSteReoLumarV12 stereo fluorescence microscope; the excitation wavelength of the green fluorescent protein was 488nm, and the emission wavelength was 507nm, and the Zeiss fluorescence inversion was used Microscope, fluorescent observation of F1 generation embryos at 12hpf, 24hpf, 48hpf, 72hpf, 4day, 5day stages, to obtain zebrafish embryos with specific expression of green fluorescent protein in the nervous system; pick out the fluorescent embryos and raise them until the zebrafish mature Fish, and further crossed with wild-type zebrafish to obtain enough transgenic zebrafish heterozygous F2; ​​heterozygous self-crossing can obtain fluorescently labeled homozygous F3.

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

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Abstract

The invention discloses a zebrafish nervous tissue-specific enhancer and cloning and application thereof and relates to the technical field of acquisition of enhancers for tissue and organ specific expressed genes. The enhancer is used for fluorescently labeling nerve cells in a zebrafish embryo development process. Sequences of the enhancer are nucleotide sequences shown in SEQ ID NO. 2. The application of the enhancer includes: developing transgenic fishes of nervous tissue-specific expressed fluorescence so as to serve for studying nerve and organ regeneration; driving specific expression of any target genes in nervous tissues so as to provide powerful tools for researchers to study development of nervous system and treatment of related diseases. The enhancer is applicable to the research on zebrafish eye development and injury regeneration processes, helps study the effect of specific genes in terms of nervous system development and controlling 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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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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