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sgRNAs, recombinant plasmids, and cell lines for upregulating expression of human Dlk1-dio3 imprinted domain non-coding RNAs

A DLK1-DIO3, recombinant plasmid technology, applied in the field of special sgRNA primer sequences

Active Publication Date: 2020-12-08
ZHEJIANG UNIV
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  • Application Information

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Problems solved by technology

In the existing studies, most of the reports suggest that the expression of non-coding RNA in the DLK1-DIO3 imprinting domain is inhibited in tumors, suggesting that it may play a tumor suppressor role

Method used

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  • sgRNAs, recombinant plasmids, and cell lines for upregulating expression of human Dlk1-dio3 imprinted domain non-coding RNAs
  • sgRNAs, recombinant plasmids, and cell lines for upregulating expression of human Dlk1-dio3 imprinted domain non-coding RNAs
  • sgRNAs, recombinant plasmids, and cell lines for upregulating expression of human Dlk1-dio3 imprinted domain non-coding RNAs

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[0027] Bladder cancer UM-UC-3 cells in good growth state were cultured. The UM-UC-3 cells were plated one day before virus infection. On the day of infection, dCAS9-VP64 lentiviral particles were added to the groups designed for the experiment to infect the target cells. Puromycine screening was performed 3 days after infection, followed by infection with sgRNA (DLK1-DIO3) lentivirus, G418 screening, and cell collection for detection or related functional studies.

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Abstract

The invention discloses sgRNA for up-regulating non-coding RNA expression in a human DLK1-DIO3 imprinted domain, a recombinant plasmid and a cell line. The sgRNA sequence is shown as SEQ ID NO. 1, andspecifically as 5'-TTTATATGGAGGCGCAGAAG-3'; the method comprises the steps of: synthesizing a nucleic acid fragment of a sgRNA sequence and inserting into a multiple cloning site of the MS2-P65-HSF1expression plasmid vector and transforming, and selecting a monoclonal strain, extracting the recombinant plasmid of MS2-P65-HSF1-sgRNA-DLK1-DIO3, and transfecting the recombinant plasmid to the target cell line which is pre-transfected with dCAS9-VP64 plasmid to obtain the cell strain for up-regulating non-coding RNA expression in the DLK1-DIO3 imprinted domain. The application can rapidly, easily and accurately up-regulate the expression of non-coding RNA on the DLK1-DIO3 imprinted domain of the cell strain.

Description

technical field [0001] The present invention relates to gene editing and its application, in particular to a dedicated sgRNA primer sequence for up-regulating the expression of non-coding RNA in the DLK1-DIO3 imprinting domain and its application. [0002] technical background [0003] Gene editing technology is a genome modification technology developed in recent years, which can perform InDel mutation, knock-in, multi-site mutation, small fragment deletion, and fragment replacement at specific sites in the genome. In the field of scientific research, gene editing technology can be used for the rapid construction of model organisms, such as the construction of specific gene knockout mice; in the field of agriculture, this technology can be used for the optimization of animal and plant varieties; It is possible to treat diseases from the source by modifying human's own genes. Therefore, gene editing technology has extremely wide application value and development prospects. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12R1/91
CPCC12N5/0693C12N15/113C12N15/63C12N2310/10
Inventor 徐鑫郑祥义王潇陈世明李江枫颜华卿
Owner ZHEJIANG UNIV
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