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United States Department of Agriculture

Agricultural Research Service

Research Project: NOVEL INTERVENTION STRATEGIES AND GENOMICS FOR CONTROLLING MASTITIS

Location: Animal Genomics and Improvement Laboratory

Title: Proinflammatory responses of a hTERT-transformed, immortalized line of cultured bovine mammary epithelial cells (BME)

Authors
item Elsasser, Theodore
item Kahl, Stanislaw
item Kerr, David -
item Zudaire, Enrique -
item Cuttitta, Frank -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 19, 2014
Publication Date: N/A

Technical Abstract: Primary cultures BME were generated from healthy mammary glands as described (Vet Immunol Immunopath 101(3-4):191-202, 2004). Towards immortalization, BME from four cows were pooled and transfected with pCI neo-hEST2-HA , a human telomerase segment containing a neomycin/Geneticin resistance selection cassette (Cell 90:785-95, 1997). Cells were grown in DMEM +10%FBS and Geneticin (800 microgram/ml) and followed through the ensuing selection growth lag to full proliferation capacity. Following 50+ passages, cells were further subcloned to increase epithelial and decrease myoepithelial cell content; the resulting culture was called ELS-321-Clone2B. For function studies and to achieve hormone and cytokine receptor access by the apical-lumenal polarized cells, cultures experiments were conducted on porous (0.4 micron) hanging well inserts coated with laminin-111. At confluence, cells had the following characteristics: tight junctions (electron microscopic confirmation of desmosomes, EpCAM-1 and E-cadherin immunostaining), expression (immunohistochemical localization ) of prolactin receptor PRLr, xanthine oxidase (XO), inducible nitric oxide synthase (iNOS), and cytokeratin-18 (<10% cells displayed myoepithelial smooth muscle actin).

Last Modified: 10/20/2014
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