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A eukaryotic expression vector expressing circular RNA

An overexpression vector, eukaryotic technology, applied in DNA/RNA fragmentation, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problem that circRNA has not been discovered and has not been studied yet, and achieves stable results and easy operation. Effect

Active Publication Date: 2020-07-03
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the emerging of this field, most circRNAs have not been discovered or studied

Method used

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  • A eukaryotic expression vector expressing circular RNA
  • A eukaryotic expression vector expressing circular RNA
  • A eukaryotic expression vector expressing circular RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, the construction of the plasmid of pcCirc

[0060] The technologies involved in the present invention are conventional technical means of molecular cloning, and the enzymes, primers, reagents and reaction conditions involved in them can be reasonably selected according to the experience of those skilled in the art unless otherwise specified, and the reagent consumables involved belong to Commercially available common products, detection means and instruments involved therein are also well known and mastered by those skilled in the art. The technical solution of the present invention will be further described through examples and test examples, but it should not be construed as a limitation of the present invention.

[0061] Taking the commercial expression vector (pcDNA3.1) as an example, we constructed the overexpression vector pcCirc, in which the sequence of the multiple cloning site region is:

[0062] CTTAAGCTTGGTACCGAGCTCGGATCCACATCGATTGGTGGAATTCTGC...

Embodiment 2

[0084] Example 2, overexpression of circRNF13 in tongue squamous cell carcinoma cells

[0085] 1. Materials and methods

[0086] 1.1 Cell culture and transfection

[0087] The tongue cancer cells Tca8113 and Cal27 in good growth state were divided into 2×10 5 Cells / well were seeded in a 6-well plate, and the 6-well plate was placed at 37°C, 5% CO2 In the incubator, the transfection of the circRNF13 overexpression vector can begin when the cells to be cultured grow to a density of 50-70%; the transfection process is as follows:

[0088] Add 100 μl of the prepared polylysine-modified silicon nanoparticle suspension carrying the circRNF13 eukaryotic expression plasmid to a sterile EP tube, and gently mix with 100 μl of serum-free medium; wash the cells 3 times with D-Hank's solution; Add 800 μl of serum-free medium (without antibiotics) to the above mixture, mix gently and add to one well of a 6-well plate; place the 6-well plate in CO 2 Incubate at 37°C for 6 hours in an incu...

Embodiment 3

[0101] Cell Culture and Transfection

[0102] Tongue squamous cell carcinoma cells Tca8113 and Cal27 in good growth state or drug-resistant cells were divided into 2×10 5 Cells / well were seeded in a 6-well plate, and the 6-well plate was placed at 37°C, 5% CO 2 In the incubator, the transfection of circRNF13 overexpression vector or siRNA can be started when the cultured cells grow to 50-70% density; the transfection process is as follows:

[0103] Add 100 μl of prepared polylysine-modified silicon nanoparticle suspension carrying circRNF13 eukaryotic expression plasmid or siRNA to a sterile EP tube, mix gently with 100 μl serum-free medium; wash cells with D-Hank's solution 3 times; add 800 μl serum-free medium (without antibiotics) to the above mixture, mix gently and add to 1 well of the 6-well plate; place the 6-well plate in CO 2 Incubate at 37°C for 6 hours in an incubator, discard the supernatant, and add complete medium to continue culturing overnight. Silicon nanop...

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Abstract

The invention discloses a eukaryotic expression vector for expressing circRNA. The eukaryotic expression vector is generally applicable to cyclization expression of various circRNAs, and the expression is efficient and stable; the expression demand of most circRNA is satisfied; with the vector for expressing circRNA, the operation is simple and feasible, and the popularization is easy. A powerfulresearch tool is provided for researching the function and the mechanism of circRNA, and theoretical support is provided for the research and development which further determine circRNA molecules as the novel marker and disease treatment target.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a eukaryotic expression vector for helping circular RNA form a circle. Background technique [0002] Human Genome Project and its follow-up DNA Elements Encyclopedia Project (The Encyclopedia of DNAElements Project, ENCODE) research results show that protein-coding gene sequences only account for 1-3% of the human genome sequence, while most of the human genome can be transcribed The sequence is non-coding RNA (non-coding RNA, ncRNA). Although non-coding RNA does not encode protein, it has been attracting much attention in the field of biomedical research due to its extensive involvement in the regulation of gene expression in cells. The most studied are linear ncRNA molecules, which can be divided into microRNA (microRNA, miRNA, 19-23nt) and long non-coding RNA (long non-coding RNA, lncRNAs, >200nt) according to the length of the RNA sequence. It plays an important role in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/113
CPCC12N15/113C12N15/66C12N15/85C12N2310/10
Inventor 熊炜郭灿熊芳曾朝阳刘凌云李勇王裕民莫勇真廖前进周钰娟李小玲李桂源
Owner CENT SOUTH UNIV
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