Protocol

Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice

This protocol was adapted from "Imaging Protein-Protein Interactions in Living Animals," Chapter 35, in Protein-Protein Interactions (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), as described below, or with tumor models grown from tumor cells stably expressing the complete reporter system. For optical imaging, the number of cells implanted can be relatively low (~1-5 x 10⁶), and imaging can begin even before the tumors are palpable. When using a regulated reporter gene, it may be necessary to perform a dose-dependent pilot experiment with the inducer or repressor before performing the primary experiments. Animal models other than mice can be used, including rats and, in theory, transgenic animals in which the reporter constructs have been stably integrated into the genome. Animals larger than the rat would be difficult to image due to poor penetration of light. For these larger-animal models, the use of other imaging technologies such as microPET should be considered.