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HPV-16-HCK cell line

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Catalogue Number 154463
Antigen/Gene or Protein Targets Used as an immortal adult human cervical keratinocyte line for pathogenesis and inflammation studies
Parental Line Human Cervical Keratinocytes
Host Human
Tissue Cervix
Disease Keywords Cancer
Relevance Contains full length HPV16 in episomal form
Production Details The 7905 base pair (bp) clone pEFHPV-16W12E (gift from Dr. Paul F. Lambert, University of Wisconsin Medical School, Madison, WI) derived from an HPV-positive patient was utilized as the HPV-16 genome for our replication assays (Flores et al., 1999). One hundred micrograms of HPV-16 genomes was digested overnight in a 750- l reaction from the pUC19 vector using the restriction enzyme BamHI followed by heat inactivation of the enzyme. The entire digested DNA was then re-ligated in a large volume of 36 ml by addition of T4 DNA ligase to the reaction mixture. This reaction was incubated overnight in a cold room (4°C) and purified using a Qiagen-tip 500 column (Qiagen DNA Maxi Kit) with buffers recommended by the manufacturer. After elution, the DNA was precipitated by addition of isopropanol and centrifugation 15,000g for 2h at 4°C. The pellet was washed with 70% ethanol and centrifuged 15,000g for an additiona l30 min at 4°C, air-dried, and resuspended in 200 l TE (10 mM Tris–Cl pH7.6 and 1 mM Na2EDTA)._x000D_Primary human cervical keratinocytes (HCKs) derived from tissue obtained from a hysterectomy performed for endometriosis were cultured on irradiated J2 3T3 fibroblast feeders in E-media (Wuetal.,1982). Cells were passaged 1:4 when they were 70 to 80% confluent. For transfections, irradiated J2 3T3 fibroblast feeders were removed differentially by short trypsin treatment, washing with PBS, and resupplementing with E media. The next day, these plates were transfected with the ligated HPV-16 episomes. For the transfections, Effectene (Qiagen) reagent was used according to the manufacturer’s instructions. Briefly,3 g of re-ligated DNA was mixed with EC buffer to a final volume of 300 l. A 29- l aliquot of Enhancer reagent was added and mixed by vortexing. The reaction mix was incubated for 5 min at room temperature and 36 l of Effectene reagen tadded and vortexed. The reaction was incubated 10 min at room temperature. Cell-culture plates were washed with 1 PBS and 7 ml of fresh media added. Three milliliters of media without serum was added to the transfection mixture, mixed gently ,and added to the plates.HCK-transfected plates were incubated at 37°C overnight in 5% CO2. The following day (day2) the media were removed from these plates and replaced with fresh E-media. Irradiated J23T3 fibroblast feeders were also added to each plate. In addition, the -galactosidase control plate was X-gal stained to control for transfection efficiency which was estimated to be from 5 to 10%. On day 3, each transfected plate was split into four 100-mm plates containing irradiated J23T3 feedercells.These plates were incubated overnight as stated above before addition of 200 g/ml G418 on day 4. Selective pressure was maintained for approximately 15–20 days before visible colonies were evident and large enough for isolation.Irradiated feeders were added weekly. Individual colonies were ring-isolated when they reached a diameter of at least 0.5cm and passaged to one well of a six-well tissue culture dish containing irradiated feeders. When they became confluent, the cells were passaged with irradiated feeders onto a 60-mm tissue culture plate and designated as passage 2 (P2). The cells were then passaged onto a 100-mm culture plate (P3) and then serially passaged 1:4 onto 100-mm culture plates using irradiated feeders._x000D_
Conditional No
Research Area Cancer, Microbiology, Virology
Growth/Phenotype Keywords The HPV-16-containing clones became immortal without a crisis and, at later passage, exhibited elevated levels of telomerase and higher levels of hTERT without any apparent increase in HPV-16 copy number, E6 transcript levels, or ability to degrade p53.
Recommended Growing Conditions G418 selection (Have with and without feeders)

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References: 1 entry

Sprague et al. 2002. Virology. 301(2):247-54. PMID: 12359427.


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References: 1 entry

Sprague et al. 2002. Virology. 301(2):247-54. PMID: 12359427.


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