Mammalian Cell Microinjection Assay to Study the Function of Rac and Rho

Microinjection has been widely used as a technique to introduce proteins and DNA into mammalian cells. A major advantage of mrcroinjection over transfection approaches IS that tt is possible to analyze very early responses to proteins; responses to microinjected proteins can be detected within minutes, and expression of protein encoded by microinjected DNA can often be detected within 2 h. In addition, most cells, including primary cells, are microinjectable, whereas many cell types are not readily transfectable. Analysts of responses m microinjected cells is usually based on immunocytochemical approaches because, in general, it is not posstble to inject sufficient numbers of cells to carry out biochemical studies. In some cases, however, microinjection has been used to analyze changes in protein phosphorylation, for example, following injection of fibroblasts with cyclic adenosme monophosphate (CAMP)-dependent protein kinase (1 ). Microinjection of DNA also provides a rapid means of assessing the localization of proteins in cells, and by tagging a protein with an epnope, it is possible to follow tts localization independently of endogenous proteins (2 ,3 ).