A clone carrier and its application for high -efficiency and stability expressed long chain non -encoding RNA

A cloning vector and overexpression technology, which is applied in the field of molecular biology, can solve the problems affecting the production process of virus genome integration and the unsuitability of lentiviral plasmids, etc., and achieve the effect of convenient function research

Active Publication Date: 2022-08-05
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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Problems solved by technology

However, based on the principle of lentiviral integration, if a termination signal appears in the middle of the LTR region, it will greatly affect the integration of the viral genome and the production process of the virus, so generally this original will not be used on the lentiviral vector, which is also a lentiviral plasmid Important reasons not suitable for expression of long non-coding RNA

Method used

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  • A clone carrier and its application for high -efficiency and stability expressed long chain non -encoding RNA
  • A clone carrier and its application for high -efficiency and stability expressed long chain non -encoding RNA
  • A clone carrier and its application for high -efficiency and stability expressed long chain non -encoding RNA

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Embodiment 1

[0038] In this example, the lncRNAs HOTAIRM1 (Gene Bank accession number: NR_038366.1) was used as the target gene, and the liver cancer cell HepG2 was used as the model to construct HepG2 cells stably overexpressed by ELECTS-HOTAIRM1, and the overexpression efficiency, gene length and oncology were verified. Function.

[0039] 1.1 Construction of ELECTS-HOTAIRM1 and PCDH-HOTAIRM1 cloning vectors

[0040] (1) Design the HOTAIRM1 cloning primer sequences HOTAIRM1-F and HOTAIRM1-R, and hand them over to Aike Biotech for synthesis. The primer sequences are as follows:

[0041] HOTAIRM1-F: 5′- CTAGCTAGCACC aaaagtttgccggcttccgcagtgat-3';

[0042] HOTAIRM1-R: 5′- ATTTGCGGCCGC caattttaatacatttattaag-3′.

[0043] Among them, the underline represents the restriction site introduced for ligation to the vector.

[0044] (2) Using the cDNA library as the template and using the HOTAIRM1 cloning primer as the PCR primer, amplify the HOTAIRM1 gene, and use the Qiagen PCR product recov...

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Abstract

The invention discloses a cloning vector for efficient and stable overexpression of long-chain non-coding RNA and its application. The cloning vector of the present invention is a double-stranded circular plasmid containing an IRDR-L-IRDR-R box, and the IRDR-L-IRDR box includes an IRDR-L sequence, a CMV promoter, a BGH poly(A) sequence, an IRDR-R sequence, PKG promoter and screening gene sequences. The cloning vector of the present invention makes the exogenous lncRNA express stably, maintains the original length, effectively maintains its functional characteristics, and more truly reflects its function.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a cloning vector for efficient and stable overexpression of long-chain non-coding RNA and its application. Background technique [0002] In the era of genomics, gene function research is an important branch of genetics. At the cellular or animal level, artificially up-regulating the expression of target genes using overexpression vectors is one of the important means to study gene function. At present, cells can be divided into transient transfection or stable transfection according to the timeliness of gene overexpression in cells. Stable transfection is the integration of exogenous genes into the genome of the cell itself. As the cells grow and divide, the exogenous genes can be stably expressed. At the same time, after antibiotic pressure screening, a cell line that can stably express proteins is finally obtained. The foreign gene does not integrate with the host cell genomic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C12N5/0693C12N5/067C12N15/113C12N2800/107C12N2800/90C12N2800/70C12N2510/00C12N2320/12C12N2330/51
Inventor 尹东张寅黄泳欣陈捷汪单兰郭雅彬
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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