Co-opting Intracellular Proteins for Cell-Specific Gene Manipulation

Abstract

Studies of complex multicellular organisms would benefit from the ability to selectively manipulate the activities of any cell type of interest. Our ability to achieve this is currently limited by technology and available resources. Here, I explore the artificial use of intracellular proteins as signals for conferring cell specificity in gene manipulation. The Green Fluorescent Protein (GFP) is a useful marker of gene expression and thousands of transgenic GFP reporter lines have been made to label different cell types, particularly in the mouse nervous system. However, the utility of transgenic GFP reporter lines is limited to labeling purposes. I exploited this resource for cell-specific gene manipulation by constructing synthetic systems that become biologically active upon interaction with GFP. Using GFP-binding nanobodies derived from Camelid antibodies, I co-opted GFP as a scaffold protein to bring together complementary split proteins that, when in a complex, can regulate processes such as transcription and DNA recombination. I demonstrate the utility of these systems for selectively manipulating GFP-expressing cells in the mouse nervous system and in zebrafish, for applications such as developmental perturbations, electrophysiology and optogenetic interrogation of neural circuits. To reduce the complexity of GFP-dependent systems, I developed a binary system in which GFP binds to a destabilized nanobody and, in doing so, stabilizes expression level of an output protein fused to the nanobody. I show that this approach could be used to construct fluorescent sensors and functional effectors that are only active in the presence of the intracellular antigen. This strategy can be extended to target a different intracellular protein, the HIV C-terminal domain (CTD), for intracellular antigen-inducible protein stabilization. The strategy used to generate CTD-inducible protein sensors may potentially be generalized to facilitate rapid design of protein-responsive sensors and effectors, based on elucidation of an amino acid code that can be applied across conserved protein scaffolds or antibody frameworks regardless of antigen identity. Thus, my work expands the experimental paradigm for manipulation of specific cell types in multicellular organisms and provides tools and approaches for increasingly precise analysis of biological processes in transgenic and wildtype animals.