Ker-CT (ATCC® CRL-4048)

Organism: Homo sapiens, human  /  Cell Type: keratinocyte  /  Tissue: foreskin  /  Disease: normal

Permits and Restrictions

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Organism Homo sapiens, human
Tissue foreskin
Cell Type keratinocyte
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 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.

Disease normal
Age neonatal
Gender male
Applications

Overexpression of CDK4 and hTERT in neonatal foreskin keratinocyte induced a dramatic upregulation of p16INK4a and milder upregualtion of p53 and p21WAF1, which became unresponsive to UV irradiation.  Despite the high levels of these checkpoint factors, Ker-CT cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify in organotypic culture RefRamirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

Karyotype Cytogenetic analysis was performed on G-banded metaphase cells from the human cell line Ker-CT and two abnormal male clones were detected. Clone 1 demonstrated trisomy 5 and trisomy 20. Clone 2 demonstrated trisomy 5 and four copies of chromosome 20.
Images Primary Keratinocytes 11 Day Differentiation Ker-CT 11 Day Differentiation
Derivation The Ker-CT cell line was immortalized by human telomerase and CDK4 using retroviral pBABE-puro hTERT and pSRalphaMSU expressing mouse CDK4.
Clinical Data male
neonatal
Antigen Expression Positive for p63 (TP63) and Cytokeratin 5 (KRT5)
Comments The Ker-CT cell line is positive for telomerase, failed to senesce, and was proliferating after more than 250 population doublings RefRamirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164  
Complete Growth Medium KGM-Gold™ BulletKit™(Lonza 00192060)
Subculturing Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation solutions for culture vessels of other sizes.
  1. Remove and discard spent medium.
  2. Briefly rinse with HBSS (ATCC 30-2213), 1 mL/25 cm2 and discard rinse solution.
  3. Add trypsin for primary cells (ATCC PCS-999-003), 1mL / 25cm2.  Place at 37oC for 4-6 minutes, until 90% of the cells have detached.  
  4. Rap flask gently to ensure cells are detached. Add 2% FBS in D-PBS, 1 mL/25cm2 to neutralize trypsin. 
  5. Centrifuge cells at 250 x g for 5 min at room temperature.
  6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
  7. Count cells, and seed 5.0 x 103 to 8.0 x 103 viable cells/cm2 to new culture vessels.

Medium Renewal: Every 2-3 days. 

As the cells become more confluent, increase the volume of media as follows: under 25% confluence feed cells 5 mL per 25 cm2, 25-45% confluence then feed cells 7.5 mL per 25 cm2, over 45% confluence then feed cells 10 mL per 25 cm2.

Cryopreservation
90% FBS, 10% DMSO
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
STR Profile Amelogenin: X,Y
CSF1PO: 7,11
D13S317: 11,13
D16S539: 9,11
D5S818: 12
D7S820: 10,11
TH01: 6,9
TPOX: 8
vWA: 15,16
Name of Depositor J Shay
References

Ramirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

Vaughan M, et al. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents. PLoS One 4(11): e7908, 2009. PubMed: 19936293

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • This material is distributed for research purposes only. A signed hTERT addendum to the ATCC Material Transfer Agreement must be sent to ATCC in advance of shipment.
  • 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
Other Documentation
Restrictions

This material requires that the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations be signed and returned to ATCC before shipment. The price listed above is for noncommercial and academic organizations only. Commercial and for-profit organizations should call for pricing.

References

Ramirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

Vaughan M, et al. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents. PLoS One 4(11): e7908, 2009. PubMed: 19936293