Nanobody-Based RFP-Dependent Cre Recombinase for Selective Anterograde Tracing in RFP-Expressing Transgenic Animals

Abstract Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. GFP-dependent gene regulation utilizes these lines to manipulate gene expression; however, its application has been limited to fluorescent proteins derived from Aequorea jellyfish. 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 (mCherry, mRFP1) are different from those for dimeric RFP (tdTomato). We confirmed target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors including Cre-DOR, target RFP, and reporter GFP vector. We found highly selective GFP expression in RFP-positive cortical neurons with 93.5 ± 0.6% of GFP-positive cells being mRFP1-positive. In estrogen receptor-beta (Esr2)-mRFP1 mice, we confirmed that Cre-DOR can be used for selective expression of membrane-bound GFP in the paraventricular nucleus of the hypothalamus. The neural projection from Esr2-expressing neurons in the hypothalamic paraventricular nucleus to the posterior pituitary was visualized by Cre-DOR. In gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we similarly achieved anterograde tracing of Grpr-expressing neurons in the medial amygdala and found that they are projecting axons to the posterior bed nucleus of the stria terminalis. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can increase or decrease 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.