Nanobody-based RFP-dependent Cre recombinase for selective anterograde tracing in RFP-expressing transgenic animals

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Abstract

<jats:title>Abstract</jats:title><jats:p>Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (<jats:italic>Esr2</jats:italic>)-mRFP1 mice and gastrin-releasing peptide receptor (<jats:italic>Grpr</jats:italic>)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.</jats:p>

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