Transduction of TAT-HA-β-galactosidase Fusion Protein into Salivary Gland-derived Cells and Organ Cultures of the Developing Gland, and into Rat Submandibular Gland in Vivo

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Abstract
We have studied the transduction of TAT-HA-β-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6–21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate β-galactosidase activity. Transduction of the fusion protein into A5 and C6–21 cells was concentration- and time-dependent. Therefore, the intensity of the β-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-β-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for β-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.