HIIF-D [R10C101-250nM] (ATCC® CRL-8200)

Organism: Cricetulus griseus, hamster, Chinese  /  Tissue: ovary  / 

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Organism Cricetulus griseus, hamster, Chinese
Tissue ovary
Product Format frozen
Culture Properties adherent
Biosafety Level 1
Gender female
Applications
in another host, produces protein interferon gamma
Derivation
The HIIF-D line was established from CHO DHFR- cells (deficient in dihydrofolate reductase) co-transformed with pSV-IFN-gamma (containing the coding sequence for human interferon gamma) and pAdD26SV(A)-3 (containing the mouse DHFR coding sequences).

Constructed by replacing the HindIII/BglII fragment containing murine Dfhr with the IFNG sequence.
The pBR322-derived sequences were also modified by replacing a EcoRI/PvuII fragment that inhibits replication in mammalian cells with an EcoRI/SalI fragment from pML.The pBR322-derived sequences were also modified by replacing a EcoRI/PvuII fragment that inhibits replication in mammalian cells with an EcoRI/SalI fragment from pML.

Also contains pAdD26SV(A)-3 which was cotransformed to express murine Dhfr. Both plasmids were digested with EcoRI before transformation.

This clone was selected after growth in 250 nM methotrexate.

Clinical Data
female
Genes Expressed
human interferon gamma
Cellular Products
human interferon gamma
Comments
The cells produce human gamma interferon.

The order of the major features in this plasmid is: SalI - SV40 promoter - HindIII - IFNG - SV40t - SV40 polyadenylation - EcoRI - ampR - ori.


Complete Growth Medium Alpha minimum essential medium without nucleosides and with 250 nM methotrexate; 90%; fetal bovine serum, 10%
Subculturing

Volumes used in this protocol are for 75 cm2 flask; 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 0.25% (w/v) Trypsin-0.53mM   EDTA solution to remove all traces of serum which  contains trypsin inhibitor.
  3. Add 2.0 to 3.0 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.

  1. Add 6.0 to 8.0 mL of complete growth medium and aspirate  cells by gently pipetting.  
  2. Add appropriate aliquots of the cell suspension to new culture vessels.
  3. Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: 2 to 3 times per week
Cryopreservation
Freeze medium: complete growth medium, 95%; DMSO, 5%
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Name of Depositor Biogen, Inc., Biogen N.V.
References

Fiers WC, Allet B. DNA sequences, recombinant DNA molecules and processes for producing human gamma interferon-like polypeptides in high yields. US Patent 5,004,689 dated Apr 2 1991

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
References

Fiers WC, Allet B. DNA sequences, recombinant DNA molecules and processes for producing human gamma interferon-like polypeptides in high yields. US Patent 5,004,689 dated Apr 2 1991