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HEK293S GnTI- (ATCC® CRL-3022™)

Organism: Homo sapiens, human  /  Cell Type: transformed with adenovirus 5 DNA  /  Tissue: embryonic kidney  / 

Permits and Restrictions

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Organism Homo sapiens, human
Tissue embryonic kidney
Cell Type transformed with adenovirus 5 DNA
Product Format frozen
Morphology epithelial-like
Culture Properties adherent
Biosafety Level 2   [Cells contain adenovirus viral DNA sequences]

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Age fetus
Applications
Widely used to overexpress various mammalian membrane proteins.
Storage Conditions liquid nitrogen vapor phase
Genes Expressed
N-acetylglucosaminyltransferase I (GnTI), not expressed
RefReeves P, et al. 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
Cellular Products
N-acetylglucosaminyltransferase I (GnTI), not expressed
RefReeves P, et al. 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
Comments

The HEK293S GnTIˉ cell line was established by methanesulfonate mutagenesis followed by Ricin selection. HEK293S GnTIˉ cells do not have N-acetyl-glucosaminyltransferase I (GnTI) activity and therefore lack complex N-glycans.RefReeves P, et al. 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   This cell line has been successfully used to overexpress a wide variety of mammalian membrane proteins. There were two versions of HEK293S GnTIˉ cells in the original publication. One was HEK293S GnTIˉ (ATCC CRL-3022), and the other version was HEK293S GnTIˉ variant constructed with tetracycline-inducible system. CRL-3022 does not contain tetracycline-inducible system or pcDNA6-TR vector.  
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%.
Subculturing
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.
  1. Remove and discard culture medium.
  2. 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.
  3. 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°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add appropriate aliquots of the cell suspension to new culture vessels.
  6. Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:5 to 1:20 is recommended.
Medium renewal: every 2 to 3 days
Cryopreservation
Freeze medium: complete growth medium, 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor P Reeves
Deposited As HEK293S GnTI-
References

Reeves P, et al. 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

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

Graham FL, et al. Defective transforming capacity of adenovirus type 5 host-range mutants. Virology 86: 10-21, 1978. PubMed: 664220

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
References

Reeves P, et al. 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

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

Graham FL, et al. Defective transforming capacity of adenovirus type 5 host-range mutants. Virology 86: 10-21, 1978. PubMed: 664220