Method for specificity overexpression of miRNA of slow virus mediated mammal cells

A mammalian and lentiviral technology, applied in the direction of retroRNA viruses, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of inability to achieve specific expression of miRNAmiRNA

Pending Publication Date: 2019-10-25
HARBIN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to solve the problem that the current miRNA expression system cannot realize the specific expression of miRNA complementation to a single miRNA, and to provide a method for specifically overexpressing miRNA in mammalian cells mediated by lentivirus

Method used

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  • Method for specificity overexpression of miRNA of slow virus mediated mammal cells
  • Method for specificity overexpression of miRNA of slow virus mediated mammal cells
  • Method for specificity overexpression of miRNA of slow virus mediated mammal cells

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specific Embodiment approach 1

[0011] Specific Embodiment 1: In this embodiment, a method of lentivirus-mediated mammalian cell-specific overexpression of miRNA is as follows: using the shRNA lentivirus expression system, the miRNA mature sequence is used as the upstream sense sequence, and the downstream and miRNA The antisense (antisense) sequence complementary to the mature sequence is mutated according to the rules of T to C and A to G, and the mutated RNA secondary neck loop structure is cloned into the shRNA lentiviral vector, and then the virus is packaged to establish a stable mutation The cell line of the RNA secondary neck loop structure; wherein 2-6 mutation sites are selected from the 2-8 bases starting from the 5' end of the antisense sequence for mutation.

[0012] Beneficial effects of this embodiment: This embodiment constructs a lentivirus-mediated method for specifically overexpressing miRNA in mammalian cells. Compared with chemical methods, it can not only overexpress a single specific mi...

specific Embodiment approach 2

[0013] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the mutated sequence needs to be predicted by software for predicting the RNA secondary neck loop structure, so as to ensure that the RNA secondary neck loop structure is not destroyed. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0014] Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: the software for predicting the RNA secondary neck loop structure is DNAMAN, RNAStructure or RNA draw. Others are the same as in the first or second embodiment.

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Abstract

The invention relates to a method for specificity overexpression of miRNA of slow virus mediated mammal cells. The method aims at solving the problem that an existing miRNA expression system cannot achieve specificity expression of single miRNA of an miRNA complementary pair. By means of an shRNA system, an miRNA mature sequence is adopted as an upstream sense sequence, an antisense sequence complementing the miRNA mature sequence at the downstream is mutated according to T to C and A to G rules, the mutated RNA second-level neck ring structure is cloned to a carrier, then virus packaging is performed, and a cell line of the RNA second-level neck ring structure obtained after stable conversion mutation is built. One specific miRNA in the complementary miRNA pair can be overexpressed, and the method is applied to the field of miRNA overexpression.

Description

technical field [0001] The invention relates to a method for specifically overexpressing miRNA in mammalian cells mediated by lentivirus. Background technique [0002] Abnormal expression of miRNA plays an important role in the occurrence and development of various human diseases. In previous studies, researchers have established many methods for specifically inhibiting miRNA maturation, such as miRNA antagonists, miRNA sponges, miRNA Strong bait etc. However, some studies have found that the significant low expression of miRNA also plays an important role in the occurrence and development of certain diseases. There are many methods suitable for miRNA overexpression, such as the use of chemically synthesized mimics or agonists and real miRNAs. The nuclear promoter initiates the expression of pri-miRNA. With the development of bioinformatics, the miRNA database has been gradually improved, and researchers have found that many endogenous mature miRNAs can completely compleme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15043
Inventor 田维明乔书培
Owner HARBIN INST OF TECH
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