A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• Fluorescent proteins like GFP are vital for cell labeling but lack inherent biological activity control.
• Existing methods for targeted gene expression often require complex genetic modifications.

Purpose of the Study:

• To engineer a versatile system for controlling gene expression in specific cells using GFP as a scaffold.
• To create a library of hybrid transcription factors responsive to GFP presence.

Main Methods:

• Co-opted GFP-binding proteins from Camelid antibodies to create GFP-based transcription factors.
• Designed modular transcription factors with tunable DNA specificity, transcriptional potency, and drug dependency.
• Utilized the system for cell-specific gene expression and functional perturbations in mouse models.

Main Results:

• Developed a library of hybrid transcription factors that induce gene expression only in the presence of GFP.
• Demonstrated GFP-controlled, cell-specific gene expression in mouse retina and brain.
• Enabled optogenetic probing of neural circuits in transgenic GFP mouse and zebrafish lines.

Conclusions:

• Established GFP as a multifunctional scaffold for creating biologically active protein complexes.
• The developed system allows for selective manipulation of diverse GFP-labeled cells across transgenic organisms.
• This approach offers a flexible platform for cell-specific gene manipulation and can be extended to other intracellular products.