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

DOI: https://doi.org/10.21203/rs.3.rs-1234462/v1

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. 

Significance Statement

Genetically encoded fluorescent proteins are widely used to observe specific cells or proteins in vivo. We generated Cre recombinase dependent on RFP (Cre-DOR) by combining RFP-specific nanobodies with split-Cre. Cre-DOR can induce specific gene expression in mRFP1-positive neurons with 93.5 ± 0.6% specificity. Using this system in Grpr-mRFP1 transgenic rats, we found specific projection from the medial amygdala to the BNST. Translocation of target RFPs greatly affected the efficiency of Cre-DOR. Using the glucocorticoid receptor, we achieved chemical upregulation of Cre-DOR activity by inducing translocation of target RFPs from the cytosol to the nucleus. Our findings extend the potential use of RFP-expressing transgenic animals and provide a unique method to utilize nuclear translocation of signal molecules for genetic manipulation.

Introduction

To understand the precise roles of specific types of neurons, their visualization and manipulation in a living animal are required. Genetically encoded fluorescent proteins are widely used to identify and observe specific types of cells in vivo. There are various fluorescent proteins, but many of them are variants of green fluorescent proteins (GFPs) derived from Aequorea jellyfish (Chalfie et al., 1994; Nagai et al., 2002; Shaner et al., 2005) or red fluorescent proteins (RFPs) from Discosoma coral (Shaner et al., 2004; Subach et al., 2009; Inoue et al., 2015). While the original coral RFP, DsRed, is obligately tetrameric (Baird et al., 2000), dimeric and monomeric variants have been created from DsRed (Campbell et al., 2002; Shaner et al., 2004). RFPs provide the inherent advantages of lower phototoxicity, lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation light (Shen et al., 2015). Therefore, various RFPs are used to visualize live cells in many transgenic RFP-expressing animals such as mice (Saederup et al., 2010; Fujihara et al., 2015), rats (Katoh et al., 2011; Uenoyama et al., 2015) and even larger animals (Chou et al., 2014; Yao et al., 2018). For selective manipulation of specific cells, driver lines expressing Cre recombinase are widely used. The combination of Cre driver mice and Cre-dependent virus vectors is an essential basis for optogenetics and chemogenetics in neuroscience. While recent genome-editing tools such as CRISPR-Cas systems have dramatically changed the generation of transgenic animals (Ran et al., 2013; Doudna and Charpentier, 2014; Heidenreich and Zhang, 2016), production and validation of a new Cre driver animal is still time-consuming, especially for large animals. Therefore, it would be useful if we could utilize existing well-characterized RFP-expressing animals to manipulate gene expression in specific cells by using Cre-dependent genetic tools.

Nanobodies are single-chain small antibodies derived from camels and they contain only one monomeric antigen-binding unit (Helma et al., 2015). Nanobodies and recombinant binders such as designed ankyrin repeat proteins (DARPins) (Binz et al., 2005; Stumpp et al., 2008) are frequently used as small molecular modules that selectively bind to target proteins. Specific recognition of target proteins by nanobodies can be utilized for not only visualization of target proteins (Irannejad et al., 2013; Yamagata and Sanes, 2018; Gerdes et al., 2020) but also protein degradation (Daniel et al., 2018), signal inhibition (Gulati et al., 2018) and property manipulation (Kirchhofer et al., 2010). Recently, GFP-dependent gene regulation methods using GFP-specific nanobodies have been reported (Tang et al., 2013; Tang et al., 2015). These techniques enable selective gene expression in GFP-expressing cells by utilizing nanobodies as specific binding modules to recognize GFPs. However, these methods have so far been limited to GFP variants and their applications to fluorescent protein-tagged endogenous proteins have not been fully studied.

In this study, we generated Cre recombinase dependent on RFPs (Cre-DOR) using nanobodies and DARPins. We found that functional binding unit pairs for monomeric RFPs (mCherry, mRFP1) are different from those for dimeric RFP (tdTomato). Using AAV vectors, we achieved highly selective expression of genes of interest in vivo. In Est2-mRFP1 mice, we confirmed the utility of Cre-DOR for selective tracing of mRFP1-expressing neurons in mRFP1-expressing transgenic animals. In Grpr-mRFP1 rats, we achieved selective labeling of Grpr-expressing neurons in the medial amygdala and detected a specific neural pathway from the posterodorsal medial amygdala (MePD) to the posterior bed nucleus of the stria terminalis (BSTp). In addition, we found that the activity of Cre-DOR is affected by cellular localization of target RFPs, and we achieved optical and pharmacological control of Cre-DOR activity by utilizing nuclear translocation of target RFPs. Our results advance genetic manipulation tools using nanobodies and DARPins utilizing existing RFP-expressing transgenic animals even if useful Cre-driver lines do not exist.

Materials And Methods

Animals

All experimental procedures with mice were approved by the Institutional Animal Experiment Committee of Jichi Medical University. Male C57BL/6J mice were purchased from Charles River Laboratories Japan (Kanagawa, Japan). The mice were maintained under a 12-hour light/dark cycle (light period: 7:30-19:30, dark period: 19:30-7:30) in a room with controlled temperature (22 ± 2℃) and humidity (55 ± 15%). Food and water were available ad libitum. Esr2-mRFP1 transgenic mice were generated by Dr. Hirotaka Sakamoto and their validation was published (Sagoshi et al., 2020). Grpr-mRFP1 transgenic rats were also generated by Dr. Hirotaka Sakamoto and their validation was published (Takanami et al., 2021). Briefly, the Grpr promoter-human heparin-binding epidermal growth factor-like growth factor-2A-mRFP1 BAC transgene was purified for microinjection using a slight modification of the procedure described previously (Abe et al., 2004). Grpr-mRFP1 transgenic rats were generated by pronuclear injection of Wistar rat embryos (Institute of Immunology Co., Ltd., Tokyo, Japan). For experiments using Esr2-mRFP1 transgenic mice and Grpr-mRFP1 transgenic rats, adult transgenic animals bred in the animal facility of Okayama University were used. All of the Grpr-mRFP1 rats and Esr2-mRFP1 mice were maintained on a 12-h light/12-h dark cycle and were provided unlimited access to water and rodent chow. The Committee for Animal Research, Okayama University, Japan authorized the experimental procedures.

Drug administration and light illumination

Dexamethasone (Dex) was purchased from Merck (Darmstadt, Germany). Dex was dissolved in ethanol as a 1 mM stock solution and diluted with culture medium to 1 µM just after plasmid transfection. An LED flat panel light (TH2-100X100BL; CCS Inc., Kyoto, Japan) was used for uniform blue light illumination (465 nm, 3.7 W/m2) of LEXY domains. The procedures for blue light stimulation were based on a previous report (Inutsuka et al., 2020). In Figure 3E and 3H, nuclei are visualized by fluorescence from Histone 2B-BFP (pAAV-EF1α-H2B-BFP-WPRE) that was co-transfected with mCherry-GR or nls-mCherry-LEXY.

DNA construction

Amino acid sequences of mCherry nanobodies (MBPs 1 - 6) were described in a previous report (Fridy et al., 2014) (originally described as LaMs 1 – 4, 6, and 8). DNA sequences of mCherry DARPins (MBP 7 and MBP 8) were described in another previous report (Brauchle et al., 2014) (originally described as 2m22 and 3m160). DNA sequences of MBPs 1 - 8 were codon-optimized, synthesized (Genscript, Piscataway, NJ), and inserted instead of GFP-specific nanobodies in pAAV-EF1α-N-CretrcintG (Addgene ID: 69570) or pAAV-EF1α-C-CreintG (Addgene ID: 69571) using NheI and EcoRI sites. Amino acid sequences of cellular localization signals were derived from previous reports (CAAX motif (Kawano et al., 2016), NES (Huang et al., 2013), and NLS (Beier et al., 2011)). GFP tagged with a palmitoylation signal (palGFP) has been used to efficiently trace neuronal fibers (Furuta et al., 2001). A vector coding palGFP was a kind gift from Dr. Takahiro Furuta (Kyoto University). DNA sequences of Histone 2B (H2B), LEXY, glucocorticoid receptor (GR), and NanoLuc were derived from (Addgene ID: 2097), (Addgene ID: 72655), (NM_001364180.2) and pNL1.1 vector (Promega, Madison, WI, USA). They were synthesized with restriction enzyme sites and inserted into pAAV-EF1α-H2B-BFP-WPRE, pAAV-CAG-FLEX-H2B-GFP-WPRE, pAAV-EF1α-NLS-mCherry-LEXY-WPRE, pAAV-EF1α-mCherry-GR-WPRE, pAAV-EF1α-EGFP-GR-WPRE and pAAV-CAG-FLEX-NanoLuc-WPRE, respectively. The FLEX switch consists of paired loxP and lox2272 sequences and enables the expression of a gene of interest only when Cre recombinase is functional (Atasoy et al., 2008).

In vitro luciferase assay

Plasmids encoding EF1α-driven fluorescent target proteins and N- and C-terminal split Cre chimeric variants were transfected by calcium phosphate into HEK293 cells (AAV293 cells purchased from Agilent Technologies, Inc., Santa Clara, CA, USA) along with plasmids encoding pAAV-CAG-FLEX-NanoLuc. Between 100 and 200 ng of total DNA was transfected into single wells of 96-well plates. Cells were ~80–100% confluent at the time of transfection. Cells were harvested one day (Figures 1 and 3) or two days (Figure 3F) later for the Nano-Glo Luciferase Assay System (Promega). All transfections were done at equal plasmid molar ratios. We used a Spark10M multimode microplate reader (TECAN, Männedorf, Switzerland) to detect luminescence.

Adeno-associated virus (AAV) production and purification

All AAV vectors were produced using the AAV Helper-Free System (Agilent Technologies, Inc., Santa Clara, CA, USA) and purified on the basis of published methods (Inutsuka et al., 2016). Briefly, HEK293 cells were transfected with a pAAV vector plasmid that included a gene of interest, pHelper and pAAV-RC (serotype 9; purchased from Penn Vector Core, Philadelphia, PA, USA) using the standard calcium phosphate method. Three days later, transfected cells were collected and suspended in artificial cerebrospinal fluid (aCSF; 124 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM KH2PO4, and 10 mM D-Glucose). After 4 freeze-thaw cycles, the cell lysate was treated with benzonase nuclease (Merck, Darmstadt, Germany) at 45°C for 15 min and centrifuged 2 times at 16,000 g for 10 min. The supernatant was used as the virus-containing solution. To measure the titer of purified virus, the supernatant was dissolved in artificial CSF. Digital PCR was performed to measure the viral titer using TaqMan MGB probes and the following primer pairs: woodchuck hepatitis virus posttranscriptional regulatory element (WPRE): 5'-VIC-CTGCTTTAATGCCTTTGTAT-MGB-3', Forward: 5'-TGCTCCTTTTACGCTATGTGGATA-3', Reverse: 5'-CATAAAGAGACAGCAACCAGGATTT-3'; human growth hormone polyA: 5'-FAM-CACAATCTTGGCTCACTG-MGB-3', Forward: 5'-GGGTCTATTGGGAACCAAGCT-3', Reverse: 5'-GGCTGAGGCAGGAGAATCG-3'. The AAV vector was stored at -80°C in small aliquots until the day of the experiment.

Stereotaxic AAV injection

Surgeries for AAV injections were conducted using a stereotaxic instrument. In Cre-DOR C57BL/6J mouse experiments (Figure 4), 600 nl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (1 x 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (1 x 1012 vg/ml), AAV9-CAG-FLEX-nlsGFP-WPRE (1 x 1012 vg/ml), and AAV9-EF1α-mRFP1-WPRE (5 x 1010 vg/ml) or AAV9-EF1α-mRuby-WPRE (5 x 1010 vg/ml) was injected in the right hemisphere M1 cortex (from bregma +0.7 mm, lateral +1.7 mm, ventral -0.8 mm) of 10-week-old male C57BL/6J mice. In Esr2-mRFP1 mouse experiments (Figure 5), 1 µl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (final concentration in the mixture: 6 x 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 x 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 x 1012 vg/ml) was injected in the paraventricular nucleus of the hypothalamus (PVN) (from bregma -0.9 mm, lateral 0.2 mm, ventral -4.3 mm) of 12-14-week-old male and female Esr2-mRFP1 transgenic or control wild-type mice. In Grpr-mRFP1 rat experiments (Figure 6), 1 µl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (final concentration in the mixture: 6 x 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 x 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 x 1012 vg/ml) was injected in the medial amygdala area (from bregma -3.0 mm, lateral ±3.5 mm, ventral -9.0 mm) of 10–15-week-old female Grpr-mRFP1 transgenic or control wild-type rats. Rat brain atlas figures were derived from Swanson, L.W. (2004) Brain maps: structure of the rat brain, 3rd edition (Creative Commons Attribution-NonCommercial 4.0 International License).

Retrograde tracing using retrobeads

Surgeries for injections of retrobeads were conducted using a stereotaxic instrument. In Grpr-mRFP1 rat experiments (Figure 5), 300 nl of Green Retrobeads™ IX (Lumafluor, Durham, NC) was injected in the BSTp (from bregma -1.1 mm, lateral 0.8 mm, ventral -7.4 mm) of 9 - 10-week-old female Grpr-mRFP1 transgenic rats. The rats were perfused one week after injection.

Immunohistochemistry and fluorescence microscopy

Three to four weeks after virus injections, mice were deeply anesthetized by Avertin and transcardially perfused with heparinized saline (20 U/ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH, 7.4). Brains were removed, post-fixed in 4% paraformaldehyde solution overnight, and transferred to 30% sucrose solution in 0.1 M phosphate buffer until they sank. Series of 40-µm-thick (Figure 4) or 30-µm-thick (Figures 5 and 6) sections were obtained with a cryostat (CryoStar NX70; Thermo Fisher Scientific, Waltham, MA, USA). For staining, coronal brain sections were immersed in a blocking buffer (10% goat serum and 0.3% Triton-X in 0.1 M PBS) and then incubated with primary antibodies at 4°C overnight. The sections were washed with the blocking buffer and then incubated with secondary antibodies for 1 hour at RT. The brain sections were mounted and examined with a fluorescence microscope (IX73, Olympus, Tokyo, Japan). Primary antibodies and secondary antibodies were diluted in the blocking buffer as follows: anti-GFP (RRID: AB591816, Medical & Biological Laboratories, Tokyo, Japan) at 1:1000 and Alexa Fluor 488 goat anti-rabbit IgG (diluted 1:1000; 1-day incubation at 4°C; A11034; Thermo Fisher Scientific). For the images shown in Figures 5 and 6, we used anti-GFP (RRID: AB1537403, Rockland Immunochemicals, Limerick, PA) at 1:2000, rabbit anti-DsRed (RRID: AB10013483, Takara Bio, Japan) at 1: 1000, Alexa Fluor 488 goat anti-chicken IgY (diluted 1:1000; 103-545-155; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 546 goat anti-rabbit IgG (diluted 1:1000; 111-546-003; Jackson ImmunoResearch Laboratories). We manually counted fluorescence-positive cells using NIH ImageJ software and calculated the relative percentage of fluorescence-positive cells. For counting in Figure 4, we analyzed one out of every four coronal brain slices.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9 for Windows (GraphPad Software, San Diego, CA). Simple comparisons of the means and SEM were performed by Student’s t-test. Multiple comparisons of the means and SEM were performed by one-way ANOVA followed by Tukey's test. A P value of less than 0.05 was considered significant in these analyses.

Results

Screening of efficient mCherry-binding protein (MBP) pairs to design Cre recombinase dependent on RFPs

First, we aimed to construct Cre recombinase dependent on RFPs based on the reported tool named Cre-DOG (Tang et al., 2015). In this system, N-terminal and C-terminal split Cre recombinase fragments are fused with specific nanobodies for target proteins, and target proteins mediate reunion of the split Cre recombinase fragments (Figure 1A). In order to identify functional pairs of binding proteins, we selected 6 nanobodies and 2 DARPins previously reported to have a highly specific binding property to a monomeric RFP, mCherry (Brauchle et al., 2014; Fridy et al., 2014), and we constructed every combination (8 + 8 = 16 constructs). The codon-optimized DNA sequences were synthesized and were inserted instead of GFP-specific nanobodies in pAAV-EF1α-N-CretrcintG (Addgene ID: 69570) or pAAV-EF1α-C-CreintG (Addgene ID: 69571) using NheI and EcoRI sites. We renamed them as MBPs (mCherry-binding proteins) 1 – 8 in this study. MBPs 1-6 are nanobodies and MBPs 7 and 8 are DARPins. As a consequence, we obtained pAAV-EF1α-N-Cre-MBP(1-8)-WPRE and pAAV-EF1α-C-Cre-MBP(1-8)-WPRE.

Then we performed in vitro luciferase reporter assays to find adequate MBP pairs that could induce reunion to reconstruct an active Cre recombinase. N-Cre-MBP, C-Cre-MBP, FLEX-NanoLuc, and target RFPs were co-transfected into HEK293 cells by the calcium phosphate method. The FLEX switch consists of paired loxP and lox2272 sequences and enables the expression of a gene of interest only when Cre recombinase is functional (Atasoy et al., 2008). NanoLuc is a small and bright luciferase from the deep-sea shrimp Oplophorus gracilirostris (Hall et al., 2012). Recombinase activities were measured as luminescence derived from the bioluminescent reaction catalyzed by NanoLuc luciferase. We tested mCherry, mRFP1, and tdTomato as target proteins. These red fluorescent proteins were all derived from the same wild-type DsRed protein (Shaner et al., 2004). While mCherry and mRFP1 are monomeric, tdTomato is a tandem dimer of two subunits. We also tested mRuby, which is a monomeric variant of the red fluorescent protein eqFP611 derived from Entacmaea quadricolor (Kredel et al., 2009), as a negative control. As shown in Figure 1B and 1C, heat maps of mCherry and mRFP1 showed similar patterns. The pair of N-Cre-MBP6 and C-Cre-MBP1 induced high activity for both mCherry and mRFP1. Twin pairs of the same MBP such as N-Cre-MBP1 and C-Cre-MBP1 or N-Cre-MBP2 and C-Cre-MBP2 showed weak reporter activities, possibly indicating competition for the same binding site by both N-Cre-MBP and C-Cre-MBP. On the other hand, the heat map for tdTomato greatly differed from those of mCherry and mRFP1 (Figure 1D). Unfortunately, we did not find any strong signal when we targeted the tetrameric RFP, DsRed (Figure 1E). The twin pair of N-Cre-MBP8 and C-Cre-MBP8 showed the highest activity for tdTomato. The heat maps for mRuby and No RFPs (Figure 1F and 1G) showed only weak recombinase activities around the maps. According to these heat maps obtained from luciferase assays, we selected the pair of N-Cre-MBP6 and C-Cre-MBP1 as a candidate pair for Cre-dependent on monomeric RFP.

Characterization of recombinase activity of Cre-DORs in vitro

Next, we investigated the recombination efficiency of the pair of N-Cre-MBP6 and C-Cre-MBP1 (Cre-DORN6C1) using a fluorescent protein reporter. Four kinds of plasmids including N-Cre-MBP6, C-Cre-MBP1, target RFPs, and FLEX-H2B-GFP were co-transfected into HEK293 cells by the calcium phosphate method (Figure 2A). H2B-GFP shows nuclear localization because H2B (histone 2B) protein binds to the DNA in the nucleus. Recombinase activities were measured as H2B-GFP expression induced by FLEX switching (Figure 2B). The fluorescent signal of GFP was enhanced by immunostaining using a GFP antibody. Quantitative cell counting of fluorescent images showed that 81.8 ± 1.5% of mCherry-positive cells were GFP-positive and that 74.1 ± 1.6% of mRFP1-positive cells were GFP-positive, while 5.6 ± 0.6% of mRuby-positive cells were GFP-positive (n = 8 each) (Figure 2C, 2D). The recombination efficiency of Cre-DORN6C1 + mCherry was 14.6-times higher than that of Cre-DORN6C1 + mRuby. The Cre-DORN6C1 system was found to be dependent on all three components of target RFPs, N-Cre-MBP6, and C-Cre-MBP1 in the system. Removal of N-Cre-MBP6 or C-Cre-MBP1 resulted in total loss of reporter activity. Cell counting showed that 0.9 ± 0.1% of mRFP1-positive cells were GFP-positive without CCre-MBP1 and that 0.8 ± 0.1% of mRFP1-positive cells were GFP-positive without NCre-MBP8 (n = 8 each). In all cases, the percentages of RFP-positive cells in GFP-positive cells were higher than 90% possibly because of the transfection method (mCherry: 98.9 ± 0.3%, mRFP1: 98.3 ± 0.3%, mRuby: 91.0 ± 1.9%, mRFP1ΔCCre: 94.4 ± 3.7%, mRFP1ΔNCre: 88.5 ± 5.6%).

We also tested the recombination efficiency of the pair of N-Cre-MBP5 and C-Cre-MBP4 (Cre-DORN5C4). Quantitative cell counting of fluorescent images showed that 57.9 ± 1.6% of mCherry-positive cells were GFP-positive and that 9.3 ± 1.1% of mRFP1-positive cells were GFP-positive, while only 0.9 ± 0.1% of mRuby-positive cells were GFP-positive (n = 8 each) (Figure 2E, 2F). The recombination efficiency of Cre-DORN5C4 + mCherry was 61.0 times-higher than that of Cre-DORN5C4 + mRuby. The Cre-DORN5C4 system was also found to be dependent on all three components of target RFPs, N-Cre-MBP5, and C-Cre-MBP4 in the system. Removal of N-Cre-MBP5 or C-Cre-MBP4 resulted in total loss of reporter activity. Cell counting showed that 0.7 ± 0.1% of mRFP1-positive cells were GFP-positive without CCre-MBP4 and that 0.8 ± 0.2% of mRFP1-positive cells were GFP-positive without NCre-MBP5 (n = 8 each). Although Cre-DORN5C4 can induce more specific recombination dependent on mCherry, we decided to use Cre-DORN6C1 in the later in vivo experiments because of its high efficiency when it was applied for mRFP1.

In the heatmap of the luciferase assay shown in Figure 1D, we found that the pair of N-Cre-MBP8 and C-Cre-MBP8 shows the highest recombination efficiency dependent on tdTomato. Therefore, we also tested the recombination efficiency of the pair of N-Cre-MBP8 and C-Cre-MBP8 (Cre-DORN8C8). Quantitative cell counting of fluorescent images showed that 46.9 ± 1.1% of tdtomato-positive cells were GFP-positive, while 12.8 ± 1.2% of mRuby-positive cells were GFP-positive (n = 4 each) (Figure 2G). Although Cre-DORN8C8 shows mild specificity against tdTomato, we thought that the S/N ratio of Cre-DORN8C8 was not sufficiently high for use in vivo.

Cellular Localization Of Target Rfps Affects Cre-dor Activity

Cre recombinase exerts its activity within the nucleus. Therefore, it is possible that cellular localization of target RFPs affects Cre-DOR recombinase activity. In order to test this hypothesis, we created mCherry and tdTomato fused with various localization signal peptides: CAAX motif for membrane localization, NES for cytosolic localization, and NLS for nuclear localization (Figure 3A). We observed clear intracellular translocalization of mRFP1 and tdTomato by being fused with these motifs. mCherry/tdTomato fused with CAAX were localized in the plasma membrane, mCherry/tdTomato fused with NES were localized in the cytosol, mCherry/tdTomato without any motif were localized in both the cytosol and nucleus, and nls-mCherry/tdTomato were localized in the nucleus (Figure 3B). Next, we performed in vitro luciferase reporter assays to investigate the effect of cellular translocation on recombinase activity. Plasmids for N-Cre-MBP, C-Cre-MBP, FLEX-NanoLuc, and target RFPs were co-transfected into HEK293 cells. When Cre-DORN6C1 or Cre-DORN8C8 targets RFPs with localization signals, the luciferase assay showed a clear difference between the cellular localizations (Figure 3C). A gradual incremental tendency of recombination activity among membrane-bound, cytosolic, and nucleic localization of target RFPs was observed. Quantitative analyses of luciferase assay data showed that recombinase activity is significantly different between Cre-DORN6C1/Cre-DORN8C8 + mCherry/tdTomato-NES and Cre-DORN6C1/Cre-DORN8C8 + nls-mCherry/tdTomato (P < 0.0001, Tukey’s multiple comparison test, n = 11). Note that average fluorescence intensity of mCherry-NES or tdTomato-NES is higher than that of nls-mCherry or nls-tdTomato.

Next, we aimed to control Cre-DOR activity by using chemical ligands. Glucocorticoid receptor (GR) is a nuclear receptor and it is translocated into the nucleus after binding its ligand, glucocorticoid (Lupien et al., 2009) (Figure 3D). Ligand-induced translocation of GR has been detected by addition of a GFP to the N-terminus of GR (Htun et al., 1996; Mikuni et al., 2007). Therefore, we attached RFPs to human glucocorticoid receptor alpha (hGRα) in order to control Cre-DOR activity. We found that mCherry-GR was localized in the cytosol without its ligand, dexamethasone (Dex) and that it translocated to the nucleus after incubation with Dex (1 µM, 1 hour) (Figure 3E). In Figure 3E, nuclei are visualized by fluorescence from Histone 2B-BFP (pAAV-EF1α-H2B-BFP-WPRE) that was co-transfected with mCherry-GR. Cre recombinase activity of Cre-DORN6C1 targeting mCherry-GR was strongly increased by the presence of Dex (Figure 3F).

An increase in Cre recombinase activity by nuclear localization of RFPs suggests that Cre-DOR activity can be manipulated by controlling the intracellular localization of target RFPs. In order to test this hypothesis, we used a light-inducible nuclear export domain called LEXY (Niopek et al., 2016). LEXY consists of an engineered LOV2 domain from Avena sativa phototropin-1 (AsLOV2), in which the C-terminal Jα helix was converted into an artificial NES. In the dark, the NES is tightly packed against the AsLOV2 core and is thus inactive. Exposure to blue light induces unfolding of the modified Jα helix, uncovering the NES (Figure 3G). We confirmed that nls-mCherry-LEXY was localized mainly in the nucleus in the dark, and blue light illumination (465 nm, 7 W/m2) induced translocation of fused RFPs into the cytosol in 30 min (Figure 3H). In Figure 3H, nuclei are visualized by fluorescence from Histone 2B-BFP that was co-transfected with nls-mCherry-LEXY. We found that blue light illumination inhibited Cre recombination activity of Cre-DORN6C1 targeting nls-mCherry-LEXY as indexed by luciferase activity (Figure 3I).

Functional assay of Cre-DOR in vivo using AAV vectors

In order to examine whether Cre-DORN6C1 functions in living animals, we generated AAV vectors encoding N-Cre-MBP6, C-Cre-MBP1, FLEX-nlsGFP, and target RFPs. GFP tagged with a nuclear localization signal (nlsGFP) is localized mainly in the nucleus. 600 nl of a mixture of virus vectors for Cre-DORN6C1, FLEX-nlsGFP (1 x 1012 vg/ml each), and target mRFP1 (5 x 1010 vg/ml) was unilaterally injected into the right side M1 cortex of wild-type 10-week-old male mice (Figure 4A). The titer of the target RFP-expressing vector was lowered to induce scattered expression and make it easy to count fluorescent protein-expressing cells separately. Recombinase activities were measured as nlsGFP expression induced by FLEX switching (Figure 4B). Four weeks after injection, mice were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP. Clear expression of nlsGFP at the injected site in the M1 cortex was observed (Figure 4C). Quantitative cell counting of fluorescent images showed that 50.2 ± 2.5% of mRFP1-positive cells were GFP-positive and that 93.5 ± 0.6% of GFP-positive cells were mRFP1-positive (n = 5 each) (Figure 4D, 4E). The expression efficiency of GFP in the center area of injection was higher than that in the peripheral area. In order to check the specificity of the Cre-DORN6C1 system in vivo, we injected vectors for Cre-DORN6C1, FLEX-nlsGFP (1 x 1012 vg/ml each), and mRuby (5 x 1010 vg/ml) as a control (Figure 4F). While we observed comparative amounts of mRuby-expressing neurons in the M1 cortex, we found only sparce nlsGFP-expressing neurons in the same area (Figure 4G). Quantitative cell counting of nlsGFP and mRuby-positive cells showed a clear difference between Cre-DORN6C1 + mRFP1-injected mice and Cre-DORN6C1 + mRuby-injected mice. The cell counting showed that 1.5 ± 0.5% of mRuby-positive cells were GFP-positive and that 13.3 ± 4.1% of GFP-positive cells were mRuby-positive (n = 5 each) (Figure 4D, 4E). The recombination efficiency of Cre-DORN6C1 + mRFP1 was 34.4-times higher than that of Cre-DORN6C1 + mRuby in these experiments. All these data suggest in vivo specificity of the Cre-DORN6C1 system using AAV vectors.

Functional Assay Of Cre-dor In Mrfp1-expressing Transgenic Mice

Next, we examined selective expression by Cre-DORN6C1 in mRFP1-expressing transgenic animals. In Esr2-mRFP1 mice, neurons in the paraventricular nucleus (PVN) are visualized by mRFP1. 1 µl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (6 x 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 x 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 x 1012 vg/ml) was injected in the PVN of Esr2-mRFP1 transgenic mice (Figure 5A). Recombinase activities were measured as expression of GFP tagged with a palmitoylation signal (palGFP) induced by FLEX switching (Figure 5B). palGFP is sorted to the plasma membrane and has been used to trace neuronal fibers anterogradely (Furuta et al., 2001; Nasanbuyan et al., 2018). Four weeks after injection, mice were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP and anti-mRFP1. Clear expression of palGFP at the injected site in the PVN was observed (Figure 5C). It has been reported that mRFP1-expressing neurons in the PVN of Esr2-mRFP1 mice include oxytocin neurons (Sagoshi et al., 2020) and oxytocin neurons send their axons into the posterior pituitary. In accordance with these previous findings, we observed clear projection from the palGFP-expressing neurons in the PVN and dense axonal terminals in the posterior pituitary (Figure 5D). Quantitative cell counting of palGFP-positive cells in the PVN showed a clear difference between mRFP(+) mice and mRFP(-) mice (Figure 5E). The cell counting showed that 24.1% of mRFP1-positive neurons in the PVN express palGFP on average (Figure 5F). These results showed the usability of Cre-DORN6C1 for detection of selective neural projection in mRFP1-expressing transgenic animals. We also confirmed that Cre-DORN6C1 can be functional in other parts of the brain such as the islands of Calleja (ICj) in Esr2-mRFP1 mice (Supplemental Figure 1).

Anterograde tracing of mRFP1-expressing neurons in Grpr-mRFP1 rats

Finally, we examined selective expression by Cre-DORN6C1 in mRFP1-expressing transgenic animals. In gastrin-releasing peptide receptor (Grpr)-mRFP1 transgenic rats, neurons in the posterior amygdala are visualized by mRFP1. 1 µl of a mixture of AAV9-EF1α-NCre-MBP6-WPRE (6 x 1012 vg/ml), AAV9-EF1α-CCre-MBP1-WPRE (6 x 1012 vg/ml) and AAV9-CAG-FLEX-palGFP-WPRE (6 x 1012 vg/ml) was injected in the medial amygdala area of male Grpr-mRFP1 transgenic rats (Figure 6A). Four weeks after injection, rats were sacrificed for immunohistochemistry and brain slices were stained with anti-GFP and anti-mRFP1. Clear and selective expression of palGFP at the injected site in the posterodorsal medial amygdala (MePD) was observed (Figure 6B). We observed clear projection from the palGFP-expressing neurons in the MePD. We found a bundle of smooth passing fibers in the stria terminalis (ste) and dense axonal terminals with varicosity in the posterior bed nucleus of the stria terminalis (BSTp or STMP) (Figure 6C). These findings suggest that mRFP-expressing neurons in the MePD send their axons to the BSTp (Figure 6E). Finally, we confirmed this neural projection using a retrograde tracer. We injected 300 nl of green retrobeads in the BSTp of Grpr-mRFP1 transgenic rats (Figure 6D). One week after injection, rats were sacrificed. We detected some mRFP1 neurons that included green retrobeads in the MePD. These results support our idea that mRFP-expressing neurons in the MePD send their axons to the BSTp (Figure 6F).

Discussion

In this study, we generated Cre recombinase dependent on RFPs (Cre-DOR) by making use of split-Cre combined with nanobodies and DARPins as binding units for target RFPs (Figure1). Cre recombinase activity was selectively induced by a monomeric RFP or by a dimeric RFP in vitro (Figure 2). The efficiency of Cre-DOR is affected by intracellular localization of target RFPs, and we achieved optical and pharmacological manipulation of Cre-DOR activity by utilizing intracellular translocation of the LINuS and glucocorticoid receptor (Figures 3). Using AAV vectors, we confirmed the efficiency and fidelity of selective induction of Cre-DOR activity in the mouse brain (Figures 4). Cre-DOR induced mRFP1-dependent expression of palGFP in Esr2-mRFP1 transgenic mice (Figures 5). We also achieved anterograde tracing of RFP-expressing neurons using Cre-DOR and found neural projection from mRFP-expressing neurons in the MePD to the BSTp in Grpr-mRFP1 transgenic rats (Figures 6).

Our results provide a new method for utilizing existing RFP transgenic animal lines to regulate gene expression selectively in RFP-expressing cells. This technique is useful not only because it can be used as a substitute of Cre transgenic lines but also because it can fundamentally provide a unique way to mark RFP-expressing cells. The number of available transgenic large animals including non-human primates has been gradually increasing (Sasaki et al., 2009; Liu et al., 2016; Yao et al., 2018). Although genome editing and other genetic techniques are being rapidly developed, it is still time-consuming to generate new transgenic animals, especially large animals. In addition, it is common to use 2A peptide for simultaneous expression of both Cre recombinase and fluorescent proteins in transgenic animals (Inutsuka et al., 2014; Hernandez et al., 2015). However, a tandem sequence linked with 2A peptide can induce unpredictable cis regulation of expression in some cases (Wang et al., 2021). Therefore, this simultaneous expression of Cre recombinase and fluorescent proteins using 2A peptide is not complete substitute for RFP-transgenic animals. Our method provides an efficient way to use the same transgenic line for different purposes.

Our findings using Cre-DOR provide valuable information on specific neural pathways in mRFP1-expressing transgenic animals. In Esr2-mRFP1 transgenic mice, we found that estrogen receptor β-expressing neurons in the PVN send their axons in the posterior pituitary (Figures 5D). We also reported that 70–80% of oxytocin neurons expressed mRFP1, while only 10% of vasopressin neurons did so in the PVN of Esr2-mRFP1 transgenic mice (Sagoshi et al., 2020). Estrogen has been found to both reduce anxiety-related behaviors and increase oxytocin peptide transcription, suggesting a role for oxytocin in this estrogen receptor β-mediated anxiolytic effect (Acevedo-Rodriguez et al., 2015). Considering that estrogen receptor α is not expressed in oxytocin neurons (Suzuki and Handa, 2005), it is reasonable to assume that estrogen receptor β in oxytocin neurons in the PVN plays a main role in this modulation. The absence of good antibodies for immunostaining for estrogen receptor β and the nuclear localization of estrogen receptor β hinder selective neural tracing of estrogen receptor β-expressing neurons in the PVN so far. Our findings provide a good way to investigate the physiological role of this specific neural pathway.

In Grpr-mRFP1 transgenic rats, we found that Grpr-expressing neurons in the MePD send their axons to the BSTp (Figures 6C). Grpr neurons play an important role in emotional responses, social interaction, and feeding behavior (Roesler and Schwartsmann, 2012). Although Grpr neurons in the lateral amygdala are frequently investigated, Grpr neurons in the medial amygdala are not well studied so far. It has been reported that the anterior and posterior medial amygdala differentially innervate downstream targets. While the anterior medial amygdala densely innervates the horizontal diagonal band of Broca and the medial olfactory tubercle, the posterior medial amygdala innervates the BNST (DiBenedictis et al., 2014). Our findings using Cre-DOR support these previous anatomical works and suggest a functional linkage between Grpr and sexual behaviors.

We generated monomeric/dimeric state-specific RFP-dependent Cre (Figure 2). Functional properties of MBP pairs seem to reflect the difference of oligomerization states among mCherry, mRFP1, tdTomato, and DsRed. Theoretically, it is reasonable to assume that pairs of identical MBPs do not work for monomeric RFPs because of binding competition on a single recognition site. Indeed, we found low luciferase activity when we used mCherry or mRFP1 as a target protein for pairs of identical MBPs (Figure 1B, 1C). However, we found that the combination of N-Cre-MBP8 and C-Cre-MBP8 is a functional pair for tdTomato. These findings show that it can be essential to prepare multiple nanobodies for designing nanobody-dependent molecular tools, while it is possible to make a Cre-DOR-like system using only one nanobody if the target proteins form homodimers within the cell. Since tdTomato is a dimeric protein, the same recognition site should be exposed at two locations in one dimer. The functional property of Cre-DORN8C8 might be utilized to detect the oligomerization state of monomeric proteins. For example, photo-induced conversion of the oligomerization state of a fluorescent protein, Dronpa, was reported (Zhou et al., 2012). Since Dronpa is also derived from its original coral tetramer protein (Ando et al., 2004), it is reasonable to establish a similar system using mCherry. Light-induced dimerization of small proteins has been reported (Imayoshi et al., 2013; Kawano et al., 2015). These systems would make it possible to achieve photo-induced switching of Cre recombinase activity.

We revealed that the Cre-DOR system is dependent on nuclear localization of target RFP proteins (Figure 3). Our findings show a limitation of Cre-DOR application in vivo. It might be difficult to use our Cre-DOR system for selective gene expression in cells expressing membrane protein fused with RFPs such as channelrhodopsin 2-mCherry or hM3Dq-mCherry in transgenic animals. On the other hand, our findings suggest that the Cre-DOR system can be used for clarification of nuclear translocation of proteins such as nuclear receptors. Cre-DORs showed minimal activity for cell membrane-localized target RFPs and high activity for nucleus-localized RFPs (Figure 3C). Therefore, it might be a good way to target membrane proteins that change their localization from the plasma membrane to the nucleus. In Notch signaling, the intercellular domain of Notch (NICD), which is a single-pass transmembrane receptor protein, is cleaved and translocates into the nucleus (Kopan and Ilagan, 2009). Amyloid beta is derived from amyloid precursor protein (APP) cleavage by γ-Secretase. Another cleavage fragment, APP intracellular domain (AICD), is also known to translocate into the nucleus, inducing the expression of related genes (Gertsik et al., 2014). With specific binding proteins, these molecules can be targeted by a similar method for specific gene manipulation of signal-on cells in the future.

In the future, it will be possible for endogenous proteins to be used as targets for split-Cre systems. Our results showed that not only nanobodies but also DARPins can be used to construct functional molecules utilizing selective binding to target proteins for reunion of split-Cre. Therefore, when multiple specific nanobodies or other binding proteins exist, it might be possible to induce selective gene expression dependent on a target endogenous protein. Indeed, we can easily find multiple promising nanobodies for endogenous proteins in an open website database (Wilton et al., 2018). In addition, a rapid approach to generate large repertoires of recombinant nanobodies and de novo proteins designed for creating customized small binding proteins have been reported (Fridy et al., 2014; Chevalier et al., 2017). GFP-dependent Flp recombinase has also been reported (Tang et al., 2016). It will be useful to employ Cre-DOR with GFP-dependent Flp recombinase at the same time to manipulate two kinds of cells in the same animal (Supplemental Figure 2).

Declarations

Acknowledgements: This work was supported by KAKENHI grants (16K08527, 16H01488, 17H06061 to A.I.; 17H04026, 17K19636, 25118008 to T.O.). A.I. was also supported by Jichi Medical University Young Investigator Award and a grant from Japan Prize Foundation. We thank Ms. Junko Kato for technical assistance. We thank Dr. T. Furuta (Kyoto University) for the generous gift of a vector coding palGFP. This work was also supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from Japan Agency for Medical Research and Development, AMED, under the grant number JP19dm0207057.

Author Contributions

A.I. and T.O. designed the experiments; A.I., S. M., H.M., R.K., R.N., K.T., and H.S. performed the experiments; A.I., S. M., H.M., R.K., R.N., K.T., and H.S. contributed to the analysis and interpretation of data; A.I. and T.O. wrote the manuscript.

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