CSF1PO: 13

D13S317: 9

D16S539: 11,12

D5S818: 12

D7S820: 10,11

THO1: 9

TPOX: 8,9

vWA: 17,18

The cells were immortalized by transduction with human papilloma virus 16 (HPV-16) E6/E7 genes.

The recombinant retrovirus vector pLXSN 16 E6/E7 containing the HPV-16 E6/E7 genes was used to transfect the ectotropic packaging cell line Psi-2.

Virus produced by the Psi-2 cells was used to infect the amphotropic packaging cell line PA317 (see ATCC CRL-9078).

Virus produced by the PA317 cells was used to transduce primary PTCs.

Although pLXSN 16 E6/E7 also confers resistance to neomycin, selection in G418 was not used to isolate transduced clones.

The cell line appears to be derived from a single cell based on Southern and FISH analysis.

The E6/E7 genes are present in the HK-2 genome as determined by PCR.

The cells retain a phenotype indicative of well differentiated PTCs.

They are positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3,beta 1 integrin, and fibronectin.

The cells are negative for factor VIII related antigen, 6.19 antigen and CALLA endopeptidase.

HK-2 cells retain functional characteristics of proximal tubular epithelium such as Na+ dependent / phlorizin sensitive sugar transport and adenylate cyclase responsiveness to parathyroid, but not to antidiuretic hormone.

The cells are capable of gluconeogenesis as evidenced by their ability to make and store glycogen.

HK-2 cells are anchorage dependent.

The cells will not grow in methylcellulose, soft agar or suspension.

HK-2 cells can reproduce experimental results obtained with freshly isolated PTCs.

• 0.05 mg/ml BPE - provided with the K-SFM kit

• 5 ng/ml EGF - provided with the K-SFM kit. NOTE: Do not filter complete medium.
  Atmosphere: air, 95%; carbon dioxide (CO2), 5%
  Temperature: 37.0°C
  Growth Conditions: Cell growth is dependent on epidermal growth factor. The cells should not be allowed to become confluent. Subculture at 80% of confluence.

1. Remove and discard culture medium.

2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.

3. 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.

4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.

5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximay 125 xg for 5 to 10 minutes.Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.

6. Incubate cultures at 37°C.