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sgRNA sequence for specifically knocking-out DHFR (Dihydrofolate Reductase) gene

A technology of dihydrofolate and reductase, applied in the field of genetic engineering, can solve problems such as high cytotoxicity and achieve the effect of increasing yield

Active Publication Date: 2017-09-26
四川丰讯科技发展有限公司
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  • 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

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  • sgRNA sequence for specifically knocking-out DHFR (Dihydrofolate Reductase) gene
  • sgRNA sequence for specifically knocking-out DHFR (Dihydrofolate Reductase) gene
  • sgRNA sequence for specifically knocking-out DHFR (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 in CHO cells

[0047] 1. Design sgRNA targeting CHO-S cell DHFR gene

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

[0049] Table 1 sgRNAs sequences

[0050] serial number

sgRNA sequence (5‐‐‐3)

direction

PAM sequence

sgRNA sequence position

sgRNA1‐h (SEQ ID NO: 1)

GCAAGAACGGAGACCUUCCC

Forward

TGG

Exon1

sgRNA2‐h (SEQ ID NO: 2)

GUCGCCGUGUCCCAGAAUAUAU

Forward

GGG

Exon1

sgRNA3‐h (SEQ ID NO: 3)

GCCAAUGCUCAGGUACUGGC

Forward

TGG

Exon1

[0051] CHO-S cell DHFR gene Exon1 partial sequence and sgRNA targeting sequence see figure 1 .

[0052]Extract the genomic DNA of CHO-S cells, use the primers in Table 2 to amplify the gen...

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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 in particular relates to a sgRNA sequence for specifically knocking out dihydrofolate reductase gene and application thereof. Background technique [0002] Chinese hamster ovary cell (CHO cell) is currently the most commonly used host cell for the production of pharmaceutical proteins, and 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 antibody, recombinant In the research and development and production of protein drugs and vaccines, it is a long-term pursuit of researchers to increase the yield of expression products in CHO cells. [0003] Dihydrofolate reductase (dihydrofolate reductase, DHFR) is a protease that catalyzes the reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, and is involved in purine metabolism in cells. When the cell line has lost the DHFR ...

Claims

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Application Information

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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
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