Inducible Calling Cards: Developing Mouse Reagents for Experimentally Controlled Transposon Insertion in vivo

Area of Science:

• Genetics
• Neuroscience
• Molecular Biology

Background:

• The piggyBac transposase facilitates genetic screens in mice.
• Fusions of piggyBac with transcription factors (TFs) create Calling Cards to mark DNA binding sites.
• Inducible transposases are needed for precise temporal control of recording in vivo.

Purpose of the Study:

• To engineer and test inducible Calling Cards (iCC) in the murine brain for drug-inducible DNA recording.
• To assess the efficacy and temporal control of Jun-iCC and Sp1-iCC systems.
• To evaluate potential developmental impacts of inducible TF-transposase fusions.

Main Methods:

• Engineered knock-in mice with tamoxifen-inducible TF-transposase fusions (Jun-iCC and Sp1-iCC).
• Tested iCC systems in vitro and in vivo for insertion efficacy and temporal control.
• Assessed mouse development, behavior, and neural activity recording.

Main Results:

• Jun-iCC mice showed tamoxifen-dependent recording and captured neural activity during seizures, but with low insertion numbers.
• Sp1-iCC resulted in higher insertion numbers but caused developmental abnormalities like reduced viability and anophthalmia.
• The ERT2 domain's interaction with Sp1 may underlie observed developmental defects.

Conclusions:

• Inducible Calling Cards enable drug-inducible transposon integration in vivo.
• Jun-iCC is suitable for recording transient neural activity, despite low insertion rates.
• Sp1-iCC requires further optimization to mitigate developmental side effects while leveraging its high insertion capacity.