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
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the current research, there is no report of knocking out the

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Experimental program
Comparison scheme
Effect test

Example Embodiment

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

[0059] 1. Knock out the FUT8 gene in CHO-S cells

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

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

[0062] Table 1 Sequence of sgRNAs

[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] Extract the genomic DNA of CHO-S cells, use the primers in Table 2 to amplify the genomic DNA, collect the amplified products from the gel, connect to the precut pUCA (LUC) vector, coat the Amp resistant plate, and pick 4 clones (labeled precut pUCA( LUC)-FUT8-...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap