Xenopus Transgenesis Using the pGateway System

Abstract

Transgenic approaches using I-SceI are powerful genome modification methods for creating heritable modifications in eukaryotic genomes. Such modifications are ideal for studying putative promoters and their temporal and spatial expression patterns in real time, in vivo. Central to this process is the initial engineering of a plasmid construct containing multiple DNA modules in a specific order prior to the integration into the target genome. One popular way of doing this is based upon the pGateway system, the modular form of which described in this chapter is known as pTransgenesis. We will initially describe the protocol of obtaining the plasmid construct containing the required sequence modules, and then the process of integrating the construct into the genome of a Xenopus embryo via co-injection with I-SceI and subsequent screening for transgenics.