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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com