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In-situ stable overexpression method of glycoprotein MUC16

An overexpression and glycoprotein technology, applied in the field of biomedicine, can solve the problem of unsuitable accommodation of target genes, and achieve the effect of long-term stable overexpression

Inactive Publication Date: 2020-12-29
云南省肿瘤医院
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Problems solved by technology

Existing plasmid vectors and lentiviral vectors are not suitable for accommodating such large target genes

Method used

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  • In-situ stable overexpression method of glycoprotein MUC16
  • In-situ stable overexpression method of glycoprotein MUC16
  • In-situ stable overexpression method of glycoprotein MUC16

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Experimental program
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Embodiment 1

[0037] 1. Basic information of vector system and sgRNA design:

[0038]MUC16 was overexpressed using the Lenti-CRISPR-dCas9 system, and three sgRNAs were used to increase the activation efficiency. The vector construction and lentiviral packaging followed the reference (Konermann S, Brigham MD, Trevino AE, etal. 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. sgRNA sequences were designed using CRISPRdirect.

[0039] Table 1. Lenti-CRISPR-dCas9 system used for MUC16 overexpression

[0040] Gene Catalog# Plasmidname MUC16overexpression Plasmid#61425 lentidCAS-VP64_Blast Plasmid#61426 lentiMS2-P65-HSF1_Hygro Plasmid#61427 lentisgRNA(MS2)_zeobackbone

[0041] Table 2. MUC16 overexpression sgRNA

[0042] Gene Seq NO. sgRNA sequence MUC16overexpression sgRNA-1 GGTTTCTAAGACATCACACA sgRNA-...

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Abstract

The invention discloses an in-situ stable overexpression method of glycoprotein MUC16, and belongs to the technical field of biological medicines. According to the invention, dCas9 is located to a promoter region of MUC16 gene on genome DNA by utilizing specific sgRNA of MUC16, and a transcription initiation complex is recruited to the greatest extent at a localization point through VP64 fused with dCas9 in combination with MS2-P65-HSF1, so that in-situ transcription activation of the MUC16 gene is effectively achieved. The lentivirus system can integrate the necessary elements for overexpression (MUC16-sgRNA, dCAS-VP64 and MS2-P65-HSF1) into genome DNA (deoxyribonucleic acid) of a cell. Along with cell division and replication, the stable overexpression of MUC16 in cell strains is achieved. According to the invention, in-situ stable overexpression of macromolecular glycoprotein MUC16 which is difficult to clone by a conventional vector is realized.

Description

technical field [0001] The invention relates to a method for in situ stable overexpression of glycoprotein MUC16, belonging to the technical field of biomedicine. Background technique [0002] In existing methods, plasmid vectors are mostly used for transient overexpression of target genes. Since plasmids cannot be integrated into cellular genomic DNA, target genes will gradually be lost with cell division. The lentiviral vector can integrate the target gene into the genomic DNA of the cell to achieve stable overexpression. Regardless of whether plasmid or viral vector is used to achieve overexpression, the target gene sequence needs to be cloned into the vector, and the expression level can be increased by increasing the gene copy number. The size of the cloned fragment is limited by the capacity of the vector. If the mRNA fragment of the target gene is too large and exceeds the capacity of the vector, it cannot be effectively constructed. For example: the maximum limit o...

Claims

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Application Information

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IPC IPC(8): C12N15/867C07K14/47
CPCC07K14/4727C07K14/4748C12N15/86C12N2740/15043C12N2800/107
Inventor 陈颖李光剑丁晓洁杨震林郭威孙远宁明杰
Owner 云南省肿瘤医院
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