INTRODUCTIONRNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for many model systems. The protocol presented here describes the cloning of a plasmid construct for use in transgenic RNAi experiments in mouse oocytes. The protocol is intended for production of a transgene by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. The procedure begins with the selection of sequences and formulation of the cloning strategy. Subsequently, the IR is cloned, inserted into the transgenic cassette, and characterized by sequencing. Finally, the transgene is released from the cassette, purified, and provided to the transgenic facility.