Stepped fine adjustment method for in-situ over-expression of long-chain non-coding RNA-LINC00842

An RNA-LINC00842, long-chain non-coding technology, which is applied in the direction of retro-transcriptional RNA virus, DNA/RNA fragments, and other methods of inserting foreign genetic materials, can solve the problem that it is difficult to achieve gradient regulation of target gene expression, and achieve long-term The effect of stabilizing overexpression

Inactive Publication Date: 2020-12-22
云南省肿瘤医院
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Problems solved by technology

The current commonly used vectors can usually achieve a relatively stable up-regulation, for example: up-regulation of about 3 times in a certain cell; but it is difficult to achieve gradient regulation of the expression of the target gene using a vector, for example: up-regulation of 2, 3, 4 times in sequence

Method used

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  • Stepped fine adjustment method for in-situ over-expression of long-chain non-coding RNA-LINC00842
  • Stepped fine adjustment method for in-situ over-expression of long-chain non-coding RNA-LINC00842
  • Stepped fine adjustment method for in-situ over-expression of long-chain non-coding RNA-LINC00842

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Embodiment 1

[0041] (1) Basic information of vector system and sgRNA design:

[0042] LINC00842 was overexpressed using the Lenti-CRISPR-dCas9 system, and three sgRNAs were used to achieve gradient regulation of expression levels. Vector construction and lentiviral packaging followed the references (Konermann S, Brigham MD, TrevinoAE, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9complex.Nature 2015;517:583–588), the Lenti-CRISPR-dCas9 system comes from Zhang Feng’s laboratory; the sgRNA sequence is designed using CRISPRdirect.

[0043] Table 1. Lenti-CRISPR-dCas9 system used for overexpression of LINC00842

[0044] Gene Plasmid Mingmu# plasmid name LINC00842 overexpression Plasmid#61425 lentidCAS-VP64_Blast Plasmid#61426 lentiMS2-P65-HSF1_Hygro Plasmid#61427 lentisgRNA(MS2)_zeobackbone

[0045] Table 2. LINC00842 overexpressed sgRNA

[0046] Gene Seq NO. sgRNA sequence LINC00842 overexpression ...

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Abstract

The invention discloses a stepped fine adjustment method for the in-situ over-expression of a long-chain non-coding RNA-LINC00842. According to the method, LINC00842 specific sgRNA is used to positiondCas9 to a promoter region of an LINC00842 gene on a genome DNA, a transcription initiation complex is recruited in different degrees at a positioning site through the binding of MS2-P65-HSF1 with dCas9-fused VP64, and thereby the gradient adjustment of the in-situ over-expression of the LINC00842 gene is effectively realized. Moreover, a lentivirus system can integrate over-expression required elements (LINC00842-sgRNA, dCAS-VP64 and MS2-P65-HSF1) into a genome DNA of cells, the stable over-expression of LINC00842 in a cell strain is realized along with cell division and replication, and finally a cell strain series for stably over-expressing the LINC00842 gene with different amplitudes is obtained.

Description

technical field [0001] The invention relates to a step fine-tuning method for in situ overexpression of long-chain non-coding RNA-LINC00842, belonging to the technical field of in situ overexpression. Background technique [0002] The above regulation and downregulation methods are very common to study gene function. Upregulation is generally achieved by overexpressing the target gene with a plasmid or viral vector. The amount of upregulation mainly depends on the method used and the upregulation range that the vector can achieve. stable amount. In fact, in the complex dynamic signaling network of cells, the direction and magnitude of gene quantitative changes will affect the state and physiological functions of cells. The current commonly used vectors can usually achieve a relatively stable up-regulation, for example: up-regulation of about 3 times in a certain cell; but it is difficult to achieve gradient regulation of the expression of the target gene using a vector, for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/90C12N15/67C12N15/113
CPCC12N15/113C12N15/67C12N15/86C12N15/907C12N2740/15043C12N2800/107C12N2310/20
Inventor 陈颖丁晓洁郭威杨震林彭静石剑林明超
Owner 云南省肿瘤医院
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