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Kit for knocking out FUT8 (alpha-1,6-fucosyltrasnferase 8) and DHFR (dihydrofolate reductase) genes in Chinese hamster ovary cells

A kit and gene technology, applied in the field of genetic engineering, can solve the problems that have not been seen, and achieve the effects of simple and easy operation, good selection of engineered cell lines, and high amplification efficiency

Active Publication Date: 2017-08-11
四川丰讯科技发展有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0006] In the current research, there is no report of knocking out the two genes of FUT8 and DHFR in CHO cells through the CRISPR-Cas9 system

Method used

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  • Kit for knocking out FUT8 (alpha-1,6-fucosyltrasnferase 8) and DHFR (dihydrofolate reductase) genes in Chinese hamster ovary cells
  • Kit for knocking out FUT8 (alpha-1,6-fucosyltrasnferase 8) and DHFR (dihydrofolate reductase) genes in Chinese hamster ovary cells
  • Kit for knocking out FUT8 (alpha-1,6-fucosyltrasnferase 8) and DHFR (dihydrofolate reductase) genes in Chinese hamster ovary cells

Examples

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Effect test

Embodiment 1

[0058] Example 1 Using the kit of the present invention to knock out the FUT8 and DHFR genes of CHO cells

[0059] 1. Knockout of the FUT8 gene in CHO-S cells

[0060] 1. Design sgRNA targeting FUT8 gene in CHO-S cells

[0061] According to the FUT8 gene sequence of CHO cells published by Genbank, sgRNA sequences targeting different exons (Exons) were designed. The designed sgRNA sequences are shown in Table 1:

[0062] Table 1 sgRNAs sequences

[0063] serial number sgRNA sequence (5‐‐‐3) direction PAM sequence sgRNA sequence position sgRNA2‐f (SEQ ID NO: 1) UGGGAUACCCACCACACUGC reverse AGG Exon8 sgRNA4‐f (SEQ ID NO: 2) ACAUGGACUCUGGGGAGAAG reverse TGG Exon9 sgRNA5‐f (SEQ ID NO: 3) GUCCAGCUUUGCAAGAAUCU reverse TGG Exon1

[0064] Genomic DNA was extracted from CHO-S cells, the genomic DNA was amplified using the primers in Table 2, the amplified product was recovered from the gel, connected to the precut pUCA (LUC)...

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Abstract

The invention provides a kit for knocking out FUT8 (alpha-1,6-fucosyltrasnferase 8) and DHFR (dihydrofolate reductase) genes in Chinese hamster ovary cells; the kit comprises sgRNA sequence described in SEQ ID NO. 1 and / or SEQ ID NO. 3, and sgRNA sequence described in SEQ ID NO. 23. The invention also provides a method for knocking out both FUT8 and DHFR genes in CHO cells and an engineering cell strain prepared by the method. The kit is suitable for quickly and efficiently knocking out FUT8 and DHFR genes in CHO cells and suitable for efficiently expressing antibody drugs with no fucose modification, provides high antibody yield and has a promising application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a kit for knocking out FUT8 gene and DHFR gene in CHO cells. Background technique [0002] Chinese hamster ovary cells (CHO cells) are currently the most commonly used host cells for the production of pharmaceutical proteins, and are widely used in the research and development and production of antibodies, recombinant protein drugs, and vaccines. Antibody drugs are currently the most important biotechnology drugs. How to further improve the efficacy and yield of antibody drugs in CHO cells is a current research hotspot. [0003] α-1,6 fucosyltransferase (α-1,6fucosyltrasnferase, FUT8) is a member of the fucosyltransferase gene superfamily, and the core fucosylation catalyzed by FUT8 is an important post-translational process for glycoproteins. modification and functional regulation. When using host cells to express antibody drugs, the glycosylation modification of the antibod...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N5/10
CPCC12N5/0682C12N9/003C12N9/1051C12N9/22C12N15/85C12N15/907C12N2800/107C12N2810/10C12Y105/01003C12Y204/01068
Inventor 罗世超罗川
Owner 四川丰讯科技发展有限公司
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