Fluorescent Calcium Indicator Protein Expression in the Brain Using Tetracycline-Responsive Transgenic Mice

Abstract

To achieve robust long-term fluorescent calcium indicator protein (FCIP) expression in mammalian neurons in vivo, classical mouse transgenesis by pronuclear DNA injection using tetracycline (Tet)-controlled genetic switches can be deployed. This protocol describes methods for regulated expression of FCIP using Tet-responsive transgenic mice. The Tet-inducible system requires three components for inducible and reversible control of gene expression: (1) a potent transcriptional activator protein, either Tet transactivator (tTA) or reverse tTA (rtTA); (2) a minimal Tet-promoter (P_tet) or a bidirectional Tet-promoter (P_tetbi) to express one or more responder genes; and (3) Tet or one of its derivatives such as doxycycline (Dox) as an inducer. To ensure a high level of FCIP expression in neurons, transgenic founder mice are screened using an ear fibroblast culture method to identify those that are responsive to Dox treatment before use in experiments. The protocol describes the use of Dox to regulate gene expression and provides a short description of in vivo recording of luciferase activity.