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Catalogue Number
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154463 |
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Antigen/Gene or Protein Targets
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Used as an immortal adult human cervical keratinocyte line for pathogenesis and inflammation studies
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Parental Line
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Human Cervical Keratinocytes
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Host
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Human
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Tissue
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Cervix
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Disease Keywords
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Cancer
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Model
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Immortalised Line
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Relevance
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Contains full length HPV16 in episomal form
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Production Details
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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_
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Conditional
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No
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Research Area
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Cancer, Microbiology, Virology
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Growth/Phenotype Keywords
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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.
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Recommended Growing Conditions
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G418 selection (Have with and without feeders)
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