[go: up one dir, main page]

WO2019096054A1 - Méthode de criblage d'une lignée cellulaire hek293 déficiente en glutamine synthétase - Google Patents

Méthode de criblage d'une lignée cellulaire hek293 déficiente en glutamine synthétase Download PDF

Info

Publication number
WO2019096054A1
WO2019096054A1 PCT/CN2018/114518 CN2018114518W WO2019096054A1 WO 2019096054 A1 WO2019096054 A1 WO 2019096054A1 CN 2018114518 W CN2018114518 W CN 2018114518W WO 2019096054 A1 WO2019096054 A1 WO 2019096054A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
screening
hek293
sequence
glutamine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/114518
Other languages
English (en)
Chinese (zh)
Inventor
薛博夫
马墨
杨银辉
白孟飞
陈莉
胡雯
钟宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eureka Biotechnology Ltd
Original Assignee
Eureka Biotechnology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eureka Biotechnology Ltd filed Critical Eureka Biotechnology Ltd
Publication of WO2019096054A1 publication Critical patent/WO2019096054A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the sgRNA sequence is one selected from the group consisting of the 5' end extension or the 3' end extension or the 1-5 base sequence of the sequence shown in SEQ ID NO. 2-82.
  • the reagents involved in the examples of the present invention are all commercially available products, which are all commercially available. All restriction enzymes were purchased from NEB; DMEM medium was purchased from Thermo; non-essential amino acid mixture was purchased from Thermo: 11140076; 0.1 mM glutamine CDM (Hyclone: SH30858.02) was purchased from Hyclone MSX was purchased from Sigma: M5379-500.
  • the PCR reaction was carried out using KAPA HiFi DNA Polymerase at an annealing temperature of 58 ° C for 15 s.
  • the recovered EGFP1 fragment and pShCMV-MCS (the MCS sequence is shown in SEQ ID NO. 83, the plasmid map is shown in Figure 13).
  • the plasmid was digested with ClaI, BamHI, purified by gel, T4 DNA ligase linkage, DH5 ⁇ competent state. Cell transformation and plasmid DNA miniprep and identification The pShCMV-EGx-MCS plasmid was constructed.
  • #2B4 and #3D6 were grown as above to a 60 mm culture dish to nearly 100% overfill.
  • the cells were scraped with a cell scraper and centrifuged to remove the supernatant. Resuspend the cells to 500 ⁇ l lysis buffer (100 mM NaCl, 10 mM Tris-Cl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS, 0.2 mg/ml proteinaseK and 100 ⁇ g/ml RNaseA) at 55 ° C incubator Incubate for 2 hours. After the reaction, the genomic DNA was purified by a phenol chloroform DNA extraction method.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une lignée cellulaire à base de cellules HEK293 déficiente en glutamine synthétase HEK293-GS-/-qui peut être passée de manière stable, adaptée à une culture en suspension, et peut être appliquée à l'expression de protéines recombinantes construite à l'aide d'un système CRISPR/Cas9. La lignée cellulaire peut exprimer diverses protéines recombinantes par criblage à l'aide d'un système de criblage GS/MSX, peut exprimer de façon stable une protéine cible après de multiples passages, et peut s'adapter à la plupart des milieux sans sérum disponibles dans le commerce.
PCT/CN2018/114518 2017-11-14 2018-11-08 Méthode de criblage d'une lignée cellulaire hek293 déficiente en glutamine synthétase Ceased WO2019096054A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711122567.6 2017-11-14
CN201711122567.6A CN107893073B (zh) 2017-11-14 2017-11-14 一种筛选谷氨酰胺合成酶缺陷型hek293细胞株的方法

Publications (1)

Publication Number Publication Date
WO2019096054A1 true WO2019096054A1 (fr) 2019-05-23

Family

ID=61805163

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/114518 Ceased WO2019096054A1 (fr) 2017-11-14 2018-11-08 Méthode de criblage d'une lignée cellulaire hek293 déficiente en glutamine synthétase

Country Status (2)

Country Link
CN (1) CN107893073B (fr)
WO (1) WO2019096054A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107893073B (zh) * 2017-11-14 2020-06-02 深圳市深研生物科技有限公司 一种筛选谷氨酰胺合成酶缺陷型hek293细胞株的方法
WO2020231943A1 (fr) 2019-05-13 2020-11-19 Dna Twopointo Inc. Modifications de cellules de mammifère à l'aide de micro-arn artificiel pour modifier leurs propriétés et compositions de leurs produits
CN110551693A (zh) * 2019-07-22 2019-12-10 佛山普津生物技术有限公司 抗生素筛选hek细胞的方法
CN113088497A (zh) * 2021-04-22 2021-07-09 河南农业大学 一种稳定敲除abhd16a基因的HEK293细胞系及其构建方法
CN114107426B (zh) * 2021-11-10 2023-01-06 苏州大学 一种筛选谷氨酰胺转运蛋白抑制剂的方法、采用该方法筛选出的抑制剂及其应用
CN116263456B (zh) * 2021-12-14 2025-10-28 中国科学院大连化学物理研究所 一种测定谷氨酰胺合成酶酶活的方法
CN117568402A (zh) * 2023-11-21 2024-02-20 上海澳斯康生物制药有限公司 一种谷氨酰胺合成酶缺陷型cho细胞系及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105523969A (zh) * 2015-12-30 2016-04-27 北京大学 抑制心肌细胞程序性坏死的CaMKII的抑制剂及用途
US9567578B1 (en) * 2016-01-04 2017-02-14 Korea Advanced Institute Of Science And Technology Cell line containing a knockout of the glutamine synthetase (GS) gene and a method of producing target proteins using a GS knockout HEK293 cell line
CN107893073A (zh) * 2017-11-14 2018-04-10 深圳市港科深研生物科技有限公司 一种筛选谷氨酰胺合成酶缺陷型hek293细胞株的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636154B (zh) * 2015-10-30 2020-09-22 中国科学院上海营养与健康研究所 sgRNA筛选系统和方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105523969A (zh) * 2015-12-30 2016-04-27 北京大学 抑制心肌细胞程序性坏死的CaMKII的抑制剂及用途
US9567578B1 (en) * 2016-01-04 2017-02-14 Korea Advanced Institute Of Science And Technology Cell line containing a knockout of the glutamine synthetase (GS) gene and a method of producing target proteins using a GS knockout HEK293 cell line
CN107893073A (zh) * 2017-11-14 2018-04-10 深圳市港科深研生物科技有限公司 一种筛选谷氨酰胺合成酶缺陷型hek293细胞株的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FAN, LIANCHUN ET AL.: "Improving the Efficiency of CHO Cell Line Generation Using Glutamine Synthetase Gene Knockout Cells", BIOTECHNOLOGY AND BIOENGINEERING, vol. 109, no. 4, 30 April 2012 (2012-04-30), XP055213290, ISSN: 0006-3592, DOI: doi:10.1002/bit.24365 *

Also Published As

Publication number Publication date
CN107893073B (zh) 2020-06-02
CN107893073A (zh) 2018-04-10

Similar Documents

Publication Publication Date Title
WO2019096054A1 (fr) Méthode de criblage d'une lignée cellulaire hek293 déficiente en glutamine synthétase
US20240368623A1 (en) New methods and systems for targeted gene manipulation
TWI747808B (zh) 新穎之ｃｈｏ整合位點及其用途
CN104379753B (zh) 位点特异性整合
KR102773555B1 (ko) 게놈 조작
US20190271041A1 (en) Epigenetic modification of mammalian genomes using targeted endonucleases
Ley et al. MAR elements and transposons for improved transgene integration and expression
CN106755091A (zh) 基因敲除载体，mh7a细胞nlrp1基因敲除方法
JP2005503778A (ja) 染色体に基くプラットホーム
WO2018227755A1 (fr) Système d'édition de base et procédé de réparation spécifique de mutations du gène hbb chez l'homme, kit de réactifs et applications correspondantes
CN114058625B (zh) 一种cho细胞基因nw_003613781.1内稳定表达蛋白质的位点及其应用
WO2019120193A1 (fr) Systèmes d'édition de gènes à base unique fragmentés et application associée
CN114085841B (zh) 一种cho细胞基因nw_003614092.1内稳定表达蛋白质的位点及其应用
JP2024099583A (ja) 安定的な標的組み込み
Xu et al. A high‐fidelity, dual site‐specific integration system in CHO cells by a Bxb1 recombinase
CN107868781A (zh) 人工合成mar片段、表达载体、表达系统及其应用
WO2023093005A1 (fr) Site capable d'exprimer de manière stable une protéine dans le gène de cellule cho nw_003613756,1 et son utilisation
WO2023093007A1 (fr) Site pour l'expression stable de la protéine dans le gène de la cellule cho nw_003614889.1, et son utilisation
CN117568402A (zh) 一种谷氨酰胺合成酶缺陷型cho细胞系及其制备方法与应用
WO2023050158A1 (fr) Procédé pour réaliser une édition sur plusieurs bases
CN119842717B (zh) 一种牛fto基因启动子突变位点及其应用
US20250320526A1 (en) Method for production of a eukaryotic host cell or cell line for lambda-integrase-mediated recombination
Balasubramanian Study of transposon-mediated cell pool and cell line generation in CHO cells
CN115725652A (zh) 一种实现多碱基编辑的方法
CN120005831A (zh) Cho整合位点及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18879303

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18879303

Country of ref document: EP

Kind code of ref document: A1