| 產品名稱 |
HIIF-D [R10C101-250nM] |
| 商品貨號 |
B164610 |
| Organism |
Cricetulus griseus, hamster, Chinese |
| Tissue |
ovary |
| Product Format |
frozen |
| Culture Properties |
adherent |
| Biosafety Level |
1
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. |
| Gender |
female |
| Applications |
in another host, produces protein interferon gamma |
| Disclosure |
This material is cited in a US or other Patent and may not be used to
infringe the claims. Depending on the wishes of the Depositor, ATCC may be
required to inform the Patent Depositor of the party to which the material was
furnished. This material may not have been produced or characterized by ATCC. |
| 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.
- Remove and discard culture medium.
- 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.
- 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.
- 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°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. |
| U.S. Patent Number |
|
| 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
|
