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
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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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  • 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

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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...

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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 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

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Application Information

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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
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