Cloning and Sequencing an Inverted Repeat

INTRODUCTION

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. The production of a transgene for transgenic RNAi in mouse can be accomplished by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. Here, we describe three different strategies that have been used successfully to clone an IR: cloning by ligating polymerase chain reaction (PCR) products, sequential cloning using a short spacer, and sequential cloning using a temporary long spacer. Once cloning has been completed, sequencing the IR is the best way to assure that both arms are sufficiently long and intact; thus, we also describe typical problems one may encounter when sequencing IRs. There are two possible strategies for sequencing a cloned IR: (1) one internal primer at the end of the IR can be used to sequence both arms in a single sequencing run or (2) external primers can be used to sequence both arms separately. Although we have successfully sequenced IRs using both strategies, we suggest using the latter to sequence transgenic constructs.