HEK293S GnTI细胞
年限： fetus
器官来源： 胚胎肾
细胞形态： 上皮样
细胞类型： 其他细胞类型
是否是肿瘤细胞： 0
物种来源： 人
ATCC Number： CRL-3022™
生长状态： 贴壁生长
数量： 大量
运输方式： 冻存运输
Designations: HEK293S GnTI-
Depositors: P Reeves
Biosafety Level: 2
Shipped: frozen
Medium & Serum: See Propagation
Growth Properties: adherent
HEK293S GnTI细胞Organism: Homo sapiens
Morphology: epithelial-like
Source: Organ: embryonic kidney
Cell Type: transformed with adenovirus 5 DNA
Cellular Products: N-acetylglucosaminyltransferase I (GnTI), not expressed [16173443]
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Isolation: chemical mutagenesis with ethyl methanesulfonate (EMS)
Age: fetus
Comments: The HEK293S GnTI- cell line was established by methanesulfonate mutagenesis followed by Ricin selection. HEK293S GnTI- cells do not have N-acetylglucosaminyltransferase I (GnTI) activity and therefore lack complex N-glycans. Subsequently, the tetracycline-inducible opsin expression system was assembled in the cell line, making it a powerful tool for expression of recombinant proteins. Using rhodopsin, the cell line was shown to add N-glycans with the structure Man5GlcNAc2 and to produce yields of up to 6mg/l. [16173443]
These cells may be grown in suspension culture in a bioreactor. HEK293S GnTI细胞[16173443]
Propagation: ATCC complete growth medium: The base medium for this cell line is ATCC-formulated DMEM:F12 Medium Catalog No. 30-2006. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Temperature: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Subculturing: Protocol: These cells adhere poorly to standard tissue culture flasks. Attachment can be enhanced by using Corning CellBIND� flasks.
Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Remove and discard culture medium.
Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
HEK293S GnTI细胞Add 1.0 to 2.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37.0°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37.0°C.
Subcultivation ratio: A subcultivation ratio of 1:5 to 1:20 is recommended.
Medium renewal: every 2 to 3 days
Preservation: Freeze medium: complete growth medium, 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Related Products: Recommended medium (without the additional serum described under ATCC Medium): ATCC 30-2006
Recommended serum: ATCC 30-2020
0.25% (w/v) Trypsin - 0.53mM EDTA in Hank's BSS (w/o Ca++, Mg ++): ATCC 30-2101
Phosphate-buffered saline: ATCC 30-2200
Cell culture tested DMSO: ATCC 4-X
References: 22282: Graham FL, et al. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36: 59-72, 1977. PubMed: 886304
22319: Graham FL, et al. Defective transforming capacity of adenovirus type 5 host-range mutants. Virology 86: 10-21, 1978. PubMed: 664220
16173443: Reeves P, et al. HEK293S GnTI细胞Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99 (21): 13419-13424, 2002. PubMed: 12370423
