Expression of Rho GTPases using adenovirus vectors

Abstract

This chapter discusses the expression of Rho guanosine 5'-triphosphates (GTPases) using adenovirus vectors. The chapter describes the techniques for construction, propagation, and use of adenovirus vectors to aid researchers interested in using adenovirus vectors to study the function of Rho GTPases. The primary advantage of adenovirus over other vectors or microinjection is that highly efficient gene transfer—greater than 90% in many cases—can be achieved in a wide range of cell types and tissues. The high efficiency of gene transfer obtained with adenovirus simplifies analysis of transgene effects as a population of cells uniformly expressing the transgene that can be obtained without the need to identify and/or sort cells based on expression of the transgene or a secondary marker gene. This allows for direct biochemical analysis of transgene effects following infection. Although somewhat more difficult to make than plasmid or retrovirus constructs, once an adenovirus construct has been established it is easier to work with than either of the aforementioned vectors. The technique for the construction of recombinant adenoviruses along with the process for large scale purification of adenovirus is also described in the chapter. The chapter concludes with a discussion of transgene expression using adenovirus vectors.