HEK293S GnTI細胞

年限： fetus

器官來源： 胚胎腎

細胞形態： 上皮樣

細胞類型： 其他細胞類型

是否是腫瘤細胞： 0

物種來源： 人

ATCC Number： CRL-3022?

生長狀態： 貼壁生長

數量： 大量

運輸方式： 凍存運輸

Designations: HEK293S GnTI-

Depositors: P Reeves

Biosafety Level: 2

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

HEK293S GnTI細胞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細胞[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細胞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細胞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