Viral Gene Transfer to Developing Mouse Salivary Glands

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have provided powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs.

Objective: Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we set out to identify new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs.

Methods: We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day (E) 12 or 13 SMGs. Recombinant AAV vectors were produced using a three- or four-plasmid procedure. All recombinant vectors used in this study encoded enhanced green fluorescent protein (eGFP), which was assayed using confocal microscopy after 48h incubation with E12 and E13 SMGs. Statistical analysis of results was performed by a two-tailed Student’s t-test.

Results: We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Using the self-complementary form of AAV2, or concurrent treatment of SMGs with pharmacologic reagents including tyrphostin 23 and epoxomicin, markedly enhanced AAV2-mediated eGFP expression. In our proof-of-principle experiment, we show that transduction of SMG epithelia with self-complementary AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis in a manner similar to the exogenous recombinant protein.

Conclusion: Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial versus mesenchymal cells in an organ undergoing branching morphogenesis.