The goal of this application is to use an in vivo retrograde ductal injection approach with the retroviral vector pLXSN16E6/E7 in isoproterenol pre-treated rats to establish immortalized cell lines of acinar origin maintaining the differentiated characteristics of their normal counterparts. Preliminary studies indicate that retrograde injection of the retroviral vector BAG produces stable integration and expression of the marker gene beta-galactosidase in acinar cells, in an isoproterenol-dependent manner. The hypothesis thus is that, following the same paradigm, acinar cells will be transduced by pLXSN16E6/E7 retrovirus and that culturing of viral protein-containing cells from injected glands will lead to the establishment of permanent cell lines with characteristics of differentiated acinar cells.
The specific aims for addressing this hypothesis are: 1) to establish optimal conditions for infection with pLXSN16E6/E7, delivered by retrograde ductal injections into salivary glands of rats treated with isoproterenol, by measuring the levels of integrated and unintegrated viral DNA, the expression of neomycin phosphotransferase mRNA using RT-PCR, and by the immunocyto-chemical demonstration of viral proteins; 2) to establish cultures and acinar cell lines from salivary glands which harbor acinar cells transduced by the viral vector; and 3) characterize the established cell lines by assessing: morphology, growth, karyotype, tumorigenic potential, and the expression of an acinar cell-specific gene, cystatin S, inducible by beta-adrenergic agonists.
| Barka, T; Gresik, E W; van Der Noen, H (2000) Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo. J Histochem Cytochem 48:1453-60 |
| Barka, T; van der Noen, H M (1997) Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo. J Histochem Cytochem 45:1533-45 |
