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

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Achieving sustained expression of fluorescent calcium indicator proteins (FCIPs) in mammalian neurons in vivo is crucial for neuroscience research.
• Classical mouse transgenesis using tetracycline (Tet)-controlled genetic switches offers a powerful tool for regulating gene expression.

Purpose of the Study:

• To describe a protocol for regulated expression of FCIPs in mammalian neurons in vivo using Tet-responsive transgenic mice.
• To outline the components and screening methods for establishing a reliable Tet-inducible system for FCIP expression.

Main Methods:

• Utilizing a Tet-inducible system comprising a transcriptional activator (tTA or rtTA), a Tet-promoter (P(tet) or P(tet)bi), and an inducer (Doxycycline/Dox).
• Screening transgenic founder mice for Dox responsiveness using ear fibroblast cultures prior to experimental use.
• Employing Dox for inducible and reversible control of gene expression and in vivo recording of luciferase activity.

Main Results:

• Established a method for robust, long-term FCIP expression in mammalian neurons in vivo.
• Demonstrated the efficacy of the Tet-inducible system for precise control over FCIP expression levels.
• Validated a screening method to ensure Dox responsiveness in transgenic mice.

Conclusions:

• The described protocol enables reliable and regulated expression of FCIPs in neurons using Tet-responsive transgenic mice.
• This method provides a valuable tool for long-term in vivo monitoring of neuronal activity.