KC-5177

CHOK1-cyno-cKIT-Cell-Line

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Background of CHOK1-cyno-cKIT-Cell-Line

This gene encodes a receptor tyrosine kinase. This gene was initially identified as a homolog of the feline sarcoma viral oncogene v-kit and is often referred to as proto-oncogene c-Kit. The canonical form of this glycosylated transmembrane protein has an N-terminal extracellular region with five immunoglobulin-like domains, a transmembrane region, and an intracellular tyrosine kinase domain at the C-terminus. Upon activation by its cytokine ligand, stem cell factor (SCF), this protein phosphorylates multiple intracellular proteins that play a role in in the proliferation, differentiation, migration and apoptosis of many cell types and thereby plays an important role in hematopoiesis, stem cell maintenance, gametogenesis, melanogenesis, and in mast cell development, migration and function. This protein can be a membrane-bound or soluble protein. Mutations in this gene are associated with gastrointestinal stromal tumors, mast cell disease, acute myelogenous leukemia, and piebaldism. Multiple transcript variants encoding different isoforms have been found for this gene.

Specifications

Catalog NumberKC-5177
Cell Line NameCHOK1-cyno-cKIT-Cell-Line
NCBI/UniProt Accession NumberXM_005555273.3
Clone Number5-4#
Host Cell LineCHOK1
DescriptionStable CHOK1 clone expressing exogenous cyno-cKIT gene.
QuantityTwo vials of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI 1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI 1640 + 10% FBS + 10μg/mL Puromycin
Selection MarkerPuromycin
MorphologyEpithelial
SubcultureSplit saturated culture 1:6-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 24 hours
Mycoplasma StatusNegative

Cell Line Generation

CHOK1-cyno-cKIT-cell-line was generated using a lentiviral vector expressing the cyno-cKIT sequence.

Characterization

Figure 1: Characterization of cyno-cKIT overexpression in the CHOK1-cyno-cKIT stable clone using FACS.

Figure 2: Characterization of cyno-cKIT in the CHOK1-cyno-cKIT stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI 1640 supplemented with 10% FBS and 10μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO2 incubator.
8. Split saturated culture 1:6-1:8 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI 1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Vilain RE, Dudding T, Braye SG, Groombridge C, Meldrum C, Spigelman AD, Ackland S, Ashman L, Scott RJ. Can a familial gastrointestinal tumour syndrome be allelic with Waardenburg syndrome? Clin Genet. 2011 Jun;79(6):554-60
2.Ahmed EA, Youssif ME. Immunohistochemical study of c-KIT (CD117) expression in renal cell carcinoma. J Egypt Natl Canc Inst. 2009 Jun;21(2):121-32.
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