Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of sgRNA sequence and its application of specific knockout dihydrofolate reductase gene

A technology of dihydrofolate and reductase, which is applied in the field of genetic engineering, can solve the problems of high cytotoxicity and achieve the effect of increasing production

Active Publication Date: 2018-04-20
四川丰讯科技发展有限公司
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing reports, only Sigma Company used ZFN technology to knock out DHFR to construct DHFR- / -type CHO cell lines, which have high cytotoxicity

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
  • A kind of sgRNA sequence and its application of specific knockout dihydrofolate reductase gene
  • A kind of sgRNA sequence and its application of specific knockout dihydrofolate reductase gene
  • A kind of sgRNA sequence and its application of specific knockout dihydrofolate reductase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Using the sgRNA sequence of the present invention to knock out the dihydrofolate reductase gene of CHO cells

[0047] 1. Design sgRNA targeting the DHFR gene of CHO-S cells

[0048] According to the DHFR gene sequence of CHO cells published by Genbank, different sgRNA sequences are designed for the first exon (Exon1). The designed sgRNA sequences are shown in Table 1:

[0049] Table 1 Sequence of sgRNAs

[0050] Serial number

SgRNA sequence (5‐‐3)

direction

PAM sequence

SgRNA sequence position

SgRNA1‐h (SEQ ID NO: 1)

GCAAGAACGGAGACCUUCCC

Positive

TGG

Exon1

SgRNA2-h (SEQ ID NO: 2)

GUCGCCGUGUCCCAGAAUAU

Positive

GGG

Exon1

SgRNA3-h (SEQ ID NO: 3)

GCCAAUGCUCAGGUACUGGC

Positive

TGG

Exon1

[0051] The DHFR gene Exon1 partial sequence and sgRNA targeting sequence of CHO-S cells can be seen figure 1 .

[0052] Extract CHO-S cell genomic DNA, use the primers in Table 2 to amplify genomic DNA, gel to recover the amplified product, and connect to the precu...

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 sgRNA sequence for specifically knocking-out a DHFR (Dihydrofolate Reductase) gene. The sequence is as shown in SEQ ID NO:3. The invention also provides the application of the sgRNA sequence and a kit for knocking-out the DHFR gene. The sgRNA which is provided by the invention can be used for knocking-out the DHFR gene in a CHO (Chinese Hamster Ovary) cell quickly, efficiency and specifically, and obtaining a DHFR gene-deleted cell line; the yield of foreign protein can be greatly improved; the application prospect is good.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a sgRNA sequence for specifically knocking out the dihydrofolate reductase gene and its application. Background technique [0002] Chinese hamster ovary cell (CHO cell) is currently the most commonly used host cell for the production of pharmaceutical proteins. Its expression products are close to natural proteins in terms of molecular structure, physical and chemical properties and biological functions, and are widely used in antibodies and recombinants. In the development and production of protein drugs and vaccines, it is a long-term pursuit of researchers to increase the output of expressed products in CHO cells. [0003] Dihydrofolate reductase (DHFR) is a protease that catalyzes the reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, and participates in the process of purine metabolism in cells. When the cell line lacks the DHFR gene, the expression vector ca...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10C12N15/53
CPCC12N5/0682C12N9/003C12N15/1137C12N15/85C12N2800/107C12N2800/80C12N2810/10C12Y105/01003
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 Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com