Efficient linking of primary DNA sequence information to gene functions in vertebrate models requires that genetic modifications and their effects are analyzed in an efficacious, controlled, and scalable manner. Thus, to facilitate analysis of gene function, new genetic tools and strategies are currently under development. Transposable elements, by virtue of their inherent ability to insert into DNA, can be developed into useful tools for chromosomal manipulations. Mutagenesis screens based on transposable elements have numerous advantages as they can be applied in vivo and are therefore phenotype-driven, and molecular analysis of the mutations is straightforward. Current progress in this field indicates that transposable elements will serve as indispensable tools in the genetic toolkit of vertebrate models. Here, we provide experimental protocols for the construction, functional testing, and application of the Sleeping Beauty transposon for insertional mutagenesis of the mouse germline.