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1. WO2020000327 - CELL LINE FOR RXRα PROTEIN STABLE HIGH EXPRESSION AND PREPARATION METHOD THEREOF

Publication Number WO/2020/000327
Publication Date 02.01.2020
International Application No. PCT/CN2018/093485
International Filing Date 28.06.2018
IPC
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
07
Animal cells or tissues
071
Vertebrate cells or tissues, e.g. human cells or tissues
079
Neural cells
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
07
Animal cells or tissues
071
Vertebrate cells or tissues, e.g. human cells or tissues
079
Neural cells
0797
Stem cells; Progenitor cells
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
09
Tumour cells
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
10
Cells modified by introduction of foreign genetic material, e.g. virus-transformed cells
12
Fused cells, e.g. hybridomas
22
Human cells
C12N 15/09 (2006.01)
C12N 5/079 (2010.01)
C12N 5/0797 (2010.01)
C12N 5/09 (2010.01)
C12N 5/22 (2006.01)
CPC
C12N 15/09
Applicants
  • 深圳市疾病预防控制中心 SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION [CN/CN]; 中国广东省深圳市 南山区龙苑路8号 No.8, Longyuan Road, Nanshan District Shenzhen, Guangdong 518055, CN
Inventors
  • 张建清 ZHANG, Jianqing; CN
  • 李云秀 LI, Yunxiu; CN
  • 蒋友胜 JIANG, Yousheng; CN
  • 吴东亭 WU, Dongting; CN
  • 彭金铃 PENG, Jinling; CN
Agents
  • 深圳市兰锋知识产权代理事务所(普通合伙) SHENZHEN LAN FENG INTELLECTUAL PROPERTY AGENCY (GENERAL PARTNERSHIP); 中国广东省深圳市 福田区园岭街道八卦三路88号荣生大厦409室 Room 409, Rongsheng Building, No.88, Bagua 3rd Road Yuanling Sub-District, Futian District Shenzhen, Guangdong 518029, CN
Priority Data
Publication Language Chinese (ZH)
Filing Language Chinese (ZH)
Designated States
Title
(EN) CELL LINE FOR RXRα PROTEIN STABLE HIGH EXPRESSION AND PREPARATION METHOD THEREOF
(FR) LIGNÉE CELLULAIRE EXPRIMANT DE MANIÈRE STABLE ET IMPORTANTE LA PROTÉINE RXRα ET SON PROCÉDÉ DE PRÉPARATION
(ZH) 一种RXRα蛋白稳定高表达细胞系及其制备方法
Abstract
(EN)
The present invention falls within the technical field of biological preparations, and particularly relates to a RXRα gene stably transfected cell line for RXRα protein stable high expression and a preparation method therefor. The cell line is a cell clone where the RXRα gene is randomly integrated into the chromosomes of an SK-N-SH cell, and the genotype for the cell line is stable and can be stably inherited with cell proliferation, and the progeny cells stably and highly express the RXRα protein, thereby greatly improving the stability of the high expression of the RXRα protein to ensure the functionality of the RXRα gene and the reliability of the tested materials. Provided is a method for preparing the cell line, comprising the following steps: designing and preparing a eukaryotic expression vector, transfecting SK-N-SH cells with the eukaryotic expression vector by means of the nuclear transfection technique, obtaining the cell clones stably transfected with the RXRα gene by means of G418 drug screening, identifying whether the cell clone is a positive cell clone that is stably transfected with the RXRα gene by means of PCR and gene sequencing, identifying the expression level of the RXRα protein in positive cloned cells by using Western blot, and then obtaining the cell line for RXRα protein stable high expression in which genotypes can be stably passaged.
(FR)
La présente invention concerne le domaine technique des préparations biologiques, et concerne en particulier une lignée cellulaire transfectée de manière stable du gène RXRα destinée à exprimer de manière importante et stable la protéine RXRα et son procédé de préparation. La lignée cellulaire est un clone cellulaire dans lequel le gène RXRα est intégré de manière aléatoire dans les chromosomes d'une cellule SK-N-SH, et le génotype de la lignée cellulaire est stable et peut être hérité de manière stable lors de la prolifération cellulaire, et les cellules de la descendance expriment de manière stable et importante la protéine RXRα, ce qui permet d'améliorer considérablement la stabilité de l'expression importante de la protéine RXRα pour assurer la fonctionnalité du gène RXRα et la fiabilité des matériels testés. L'invention concerne un procédé de préparation de la lignée cellulaire, comprenant les étapes suivantes : concevoir et préparer un vecteur d'expression eucaryote, transfecter des cellules SK-N-SH avec le vecteur d'expression eucaryote à l'aide de la technique de transfection nucléaire, obtenir des clones cellulaires transfectés de manière stable avec le gène RXRα à l'aide d'un criblage avec le médicament G418, identifier si le clone cellulaire est un clone de cellule positif qui est transfecté de manière stable avec le gène RXRα à l'aide d'une PCR et d'un séquençage génique, identifier le niveau d'expression de la protéine RXRα dans des cellules clonées positives en utilisant un transfert Western, puis obtenir la lignée cellulaire exprimant de manière stable et importante la protéine RXRα dans laquelle des génotypes peuvent être passés de manière stable.
(ZH)
本发明属于生物制备技术领域,特别涉及一种RXRα蛋白稳定高表达的RXRα基因稳定转染细胞系及其制备方法。所述细胞系为RXRα基因随机整合至SK-N-SH细胞染色体的细胞克隆,细胞系基因型稳定,可随细胞增殖稳定遗传,子代细胞内稳定高表达RXRα蛋白,大大提升了RXRα蛋白高表达的稳定性,以保证RXRα基因功能性和受试材料的可靠性。本发明提供了一种所述细胞系的制备方法,设计制备真核表达载体,经细胞核转染技术以真核表达载体转染SK-N-SH细胞,经G418药物筛选得到RXRα基因稳定转染成功的细胞克隆,PCR、基因测序鉴定其是否为RXRα基因稳定转染阳性的细胞克隆,采用Western blot技术鉴定阳性克隆细胞内RXRα蛋白表达水平,从而得到基因型可稳定传代的RXRα蛋白稳定高表达细胞系。
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