The mammalian central nervous system (CNS) contains many hundreds of molecularly and functionally distinct cell types, which comprise the basic building blocks of neural circuitry. Individual cell types can be labeled and manipulated using transgenic and knock-in animals, but this approach, is slow, expensive, and limited in scope. Furthermore, it cannot be applied to higher primates or humans. We propose to develop an approach that will allow the selective targeting of individual CNS cell types in wildtype individuals, from a range of mammalian species. This is a modification of a recently developed technology known as CRE-DOG that uses pairs of camelid nanobdies to scaffold assembly of functional split Cre recombinase in the presence of GFP. We propose to use this general approach to target endogenous cell subtype-specific transcription factors using Fn3-based recombinant monobodies, which can be rapidly produced and screened in vitro, and use these to induce assembly of split Cre and Dre recombinase. These reagents can then be used to induce cell-specific activation of expression of reporter and effector constructs delivered by electroporation or viral vector. As proof of principle for this approach, we will first use Fn3-basd pairs of anti- GFP monobodies to scaffold assembly of split Cre and Dre in vivo. We will next raise pairs of monobodies against cell-specific retinal transcription factors, and demonstrate that these can scaffold assembly of functional Cre recombinase, and develop expression constructs that allow Cre-dependent expression of these reagents to avoid potential disruptive effects of monobody expression. Following this, we will demonstrate that these reagents direct cell-specific Cre activation in neonatal retina. Finally, if proven successful, we will generate a toolbx of reagents that will enable selective activation of reporter and effector constructs in the major cell types of retina and cerebral cortex, in both mice and humans.
Cell-specific labeling in the CNS using transgenesis is a powerful technique, but also slow, expensive and not suitable for many model organisms. We propose an alternative approach that uses recombinant single-chain antibodies targeting evolutionarily conserved epitopes on cell-specific transcription factors to scaffold assembly of active Cre and Dre recombinase. This technique has the potential to allow simultaneous labeling of multiple CNS cell types in both mice and humans.
| Sha, Fern; Salzman, Gabriel; Gupta, Ankit et al. (2017) Monobodies and other synthetic binding proteins for expanding protein science. Protein Sci 26:910-924 |
