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Cloning vector for efficient and stable over-expression of long chain non-coding RNA, and application of cloning vector

A cloning vector and overexpression technology, applied in the field of molecular biology, can solve problems such as inappropriate lentiviral plasmids and affecting the production process of viral genome integration

Active Publication Date: 2020-10-20
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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  • Cloning vector for efficient and stable over-expression of long chain non-coding RNA, and application of cloning vector
  • Cloning vector for efficient and stable over-expression of long chain non-coding RNA, and application of cloning vector
  • Cloning vector for efficient and stable over-expression of long chain non-coding RNA, and application of cloning vector

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Embodiment 1

[0038] In this example, using the lncRNAs HOTAIRM1 (Gene Bank accession number: NR_038366.1) as the target gene and the liver cancer cell HepG2 as a model, HepG2 cells with stable overexpression of ELECTS-HOTAIRM1 were constructed, 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, which will be synthesized by Aiji Biotechnology Co., Ltd. The primer sequences are as follows:

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

[0042] HOTAIRM1-R: 5′- ATTTGCGGCCGC caatttta at catttattaag-3′.

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

[0044] (2) Using the cDNA library as a template, HOTAIRM1 gene was amplified using HOTAIRM1 cloning primers as PCR primers, and the PCR product was purified...

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Abstract

The invention discloses a cloning vector for the efficient and stable over-expression of long chain non-coding RNA, and an application of the cloning vector. The cloning vector of the invention is a double-stranded circular plasmid containing an IRDR-L-IRDR-R box, and the IRDR-L-IRDR-R box comprises an IRDR-L sequence, a CMV promoter, a BGH poly(A) sequence, an IRDR-R sequence, a PKG promoter anda screening gene sequence. The cloning vector of the invention can stably express exogenous lncRNA, keep the original length, effectively maintain the functional characteristic of the exogenous lncRNA, and more truly reflect the function of the exogenous lncRNA.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a cloning vector for efficiently and stably overexpressing long-chain non-coding RNA and its application. Background technique [0002] In the era of genomics, the study of gene function is an important branch of genetics. At the cell or animal level, using overexpression vectors to artificially up-regulate the expression of target genes is one of the important means to study gene functions. At present, cells can be divided into transient strains or stable strains according to the timeliness of gene overexpression in cells. Stable transfection is the integration of exogenous genes into the cell's own genome. As the cells grow and divide, exogenous genes can be stably expressed. At the same time, they are screened under pressure with antibiotics to finally obtain cell lines that can stably express proteins. The exogenous gene and host cell genomic DNA did not integrate in the tran...

Claims

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Application Information

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Patent Type & Authority Applications(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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