Adenoviral-mediated gene transfer in two-dimensional and three-dimensional cultures of mammary epithelial cells.

Publisher Summary To overcome problems associated with gene transfer into primary cultures, a technique is developed in this chapter using adenovirus that allows expressing heterologous genes with 75–95% efficiency in primary mammary epithelial cells cultured in a three-dimensional matrix. This provides a new source of molecular tools to dissect signaling and phenotypic regulation. The chapter describes how to isolate primary mammary epithelial cells, how to culture them in three-dimensional matrices, and how to obtain high levels of heterologous gene expression. Recombinant adenoviruses have become a very attractive and versatile vector system for the transfer of genes into mammalian cells and are currently used for a variety of purposes, including gene transfer in vitro , vaccination in vivo and gene therapy. The chapter discusses the pAdEasy system to generate recombinant adenoviruses. The system uses the major immediate early human cytomegalovirus (CMV) promoter to drive expression of the transgene, which has been found to induce extremely high-levels of expression and is an effective promoter in differentiated mammary epithelial cells.