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

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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
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gene
screening
hek293
sequence
glutamine
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PCT/CN2018/114518
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Chinese (zh)
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薛博夫
马墨
杨银辉
白孟飞
陈莉
胡雯
钟宇
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深圳市深研生物科技有限公司
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Publication of WO2019096054A1 publication Critical patent/WO2019096054A1/fr

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    • 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
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    • 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
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    • 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
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    • 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 RNAses, DNAses
    • 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
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    • 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.

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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 WO2019096054A1 (fr)

Applications Claiming Priority (2)

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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

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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
AU2020274339C1 (en) 2019-05-13 2023-08-31 Dna Twopointo Inc. Modifications of mammalian cells using artificial micro-RNA to alter their properties and the compositions of their products
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
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

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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)

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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 *

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