In this post-genomic era, emphasis has shifted from identifying genes to understanding the physiological functions of gene products and their implications in human diseases. The use of transgenic mice is one of the key approaches in elucidating gene function and regulation. Transgenic mice have wide applications in biomedical research. These include (1) gain-of-function studies by overexpressing a protein in a tissue-specific manner; (2) loss-of-function studies by overexpressing a dominant-negative construct; (3) mapping functional domains by expressing various mutated constructs in a null (knockout mice) background; (4) mapping regulatory elements using a reporter gene; and (5) labeling subsets of cells with fluorescent proteins for live in vivo imaging of cell structure and function. In this chapter, we provide overviews as well as detailed protocols for each step involved in the generation of transgenic mice, including the selection of regulatory elements, purification of DNA, obtaining fertilized eggs, pronuclear injection, and genotyping of transgenic mice.