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Genetic targeting of chemical indicators in vivo

Nature Methods volume 12, pages 137–139 (2015)Cite this article

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Abstract

Fluorescent protein reporters have become the mainstay for tracing cellular circuitry in vivo but are limited in their versatility. Here we generated Cre-dependent reporter mice expressing the Snap-tag to target synthetic indicators to cells. Snap-tag labeling worked efficiently and selectively in vivo, allowing for both the manipulation of behavior and monitoring of cellular fluorescence from the same reporter.

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Figure 1: Ex vivo and in vivo Snap-tag labeling.
Figure 2: In vivo Snap-tag–mediated axonal ablation.

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Acknowledgements

This study was technically supported by EMBL Monterotondo Transgenic facility and EMBL Monterotondo Microscopy facility. We thank V. Paribeni for mouse husbandry.

Author information

Author notes

  1. Guoying Yang and Fernanda de Castro Reis: These authors contributed equally to this work.

Authors and Affiliations

  1. Mouse Biology Unit, European Molecular Biology Laboratory (EMBL), Monterotondo, Italy

    Guoying Yang, Fernanda de Castro Reis, Mayya Sundukova, Sofia Pimpinella, Antonino Asaro, Laura Castaldi, Laura Batti, Daniel Bilbao & Paul A Heppenstall

  2. Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany

    Guoying Yang & Paul A Heppenstall

  3. Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Luc Reymond & Kai Johnsson

  4. National Centre of Competence of Research in Chemical Biology, Lausanne, Switzerland

    Luc Reymond & Kai Johnsson

Authors
  1. Guoying Yang
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  2. Fernanda de Castro Reis
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  3. Mayya Sundukova
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  11. Paul A Heppenstall
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Contributions

L.R., K.J. and P.A.H. devised the study. G.Y., F.d.C.R., K.J. and P.A.H. contributed to manuscript writing. G.Y., F.d.C.R., M.S., S.P., A.A., L.C., L.B., D.B. and P.A.H. performed experiments.

Corresponding author

Correspondence to Paul A Heppenstall.

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Competing interests

K.J. has filed patents on Snap-tag and SiR-Snap.

Integrated supplementary information

Supplementary Figure 1 Generation of SnapCaaX lines.

(a) Schematic diagram of the wild type Rosa26 locus. (b) The Ai strategy and its recombination product. Cre-mediated removal of the stop cassette induces expression. Also included are PhiC31 recognition sites AttB/AttP for removal of the PGK-Neo, and FRT sites to allow for Flp recombinase–mediated replacement as described for the original Ai9 vector. (c) The FLEx strategy and its recombination product. Cre-mediated recombination induces inversion and then excision of a stop cassette (d) Southern blot of positive and negative ES clones targeted using the Ai strategy. (e) Southern blot of positive and negative ES clones targeted with the FLEx strategy. (f) SNAPCaaX transcript levels in the Ai and FLEx targeting strategies. (g) In-gel fluorescence of protein extracts labelled with 505-Star. Lane 1, Control. Lane 2, FLEx. Lane 3, Ai. A western blot of actin as loading control is shown below.

Supplementary Figure 2 Deleter-Cre induces robust expression of membrane-associated Snap-tag.

(a) Skin was isolated from mouse tail, labeled with SiR-SNAP for 30 minutes in PBS, fixed with 4%PFA for 20min, washed with PBS and then stained with Hoechst before confocal imaging. Negative control is shown in the lower panel. Scale bar, 20μm. (b) Brains were sectioned by microtome at a thickness of 300μm. Slices were labeled with SiR-SNAP in culture medium overnight, washed in PBS and fixed with 4%PFA for 20 minutes, washed in PBS again and stained with Hoechst before confocal image. Cortex is shown in the upper panel and negative control in the lower panel. Scale bar, 20μm. (c) Liver was sectioned by microtome at a thickness of 200μm. Slices were labeled with SiR-SNAP in PBS for 30min, fixed with 4%PFA for 20min, washed in PBS and stained with Hoechst before confocal imaging. Negative control is shown in the lower panel. Scale bar, 100μm. (d) Whole mount DRG were labeled with SiR-SNAP in culture medium overnight, washed in PBS and fixed with 4%PFA for 20min. Negative control is shown in the right panel. Scale bar, 100μm.

Supplementary Figure 3 Ex vivo Snap labeling and controls.

(a) DRG sections from Avil-Cre::SNAPCaaX mice labelled with TMR-Star and neuronal marker PGP9.5. (b) Whole mount DRG labeled with TMR-Star. Upper panels Avil-Cre::SNAPCaaX, lower panels control SNAPCaaX mice. (c) E13.5 embryos from Vglut2-Cre::SNAPCaaX (left) and control SNAPCaaX mice (center) labeled with TMR-Star, fixed in methanol/acetone and imaged using stereo microscopy. Circuitry innervating the developing whisker follicles is shown in the right panel. (d and e) Negative staining for SNAP in skin (d) and brain (e) from control SNAPCaaX mice. (f and g) 500μm spinal cord slices from Avil-Cre::Rosa26SNAPCaaX mice labeled with TMR-Star (f) and from control SNAPCaaX mice (g). (h and i) Higher magnification of TMR-Star labelling in cerebellum (h) and hippocampus (i) from NFH-Cre::SNAPCaaX or control SNAPCaaX mice. Images were over-exposed to allow visualization of cell bodies. All scale bars 100μm, except (e, f and g), 400μm.

Supplementary Figure 4 Optical clearing of SNap-labeled tissue.

(a-e) TMR-Star labelled DRG from Avil-Cre::SNAPCaaX mice cleared with BAAB (a), DBE (b), methyl salicylate (c), SeeDB (d), or Scale (e). (f) TMR-Star labelled skin cleared with methylsalicylate. (g) TMR-Star labelled 1mm spinal cord slice cleared with BAAB. Scale bar 50μm.

Supplementary Figure 5 In vivo Snap-tag labeling controls and time course.

(a-c) Negative controls for live tissue labelling in live hairy skin (a), scale bar 50μm, eye(b), scale bar 1mm, and cornea (c), scale bar 100um, from control mice labelled with SiR-SNAP(a),(b) and TMR-Star(c). (d-f). Time course of SiR-SNAP labelling in skin of Avil-Cre::SNAPCaaX mice at 4 (d), 8 (e), and 24 (f) hours after injection. Scale bar 50μm.

Supplementary Figure 6 Cellular ablation using the Snap-tag.

(a) Flow cytometry analysis of cell viability in HEK293 cells transfected with SNAPCaaX, labelled with BG-fluorescein or 505-star, and illuminated. (b) Propidium iodide labelling of SNAPCaaX transfected HEK293 cells labelled with BG-Fluorescein and illuminated (c) Propidium iodide labelling of SNAPCaaX transfected HEK293 cells labelled with 505-Star and illuminated. (d) Propidium iodide labelling of control HEK293 cells labelled with BG-Fluorescein and illuminated.

Supplementary Figure 7 Selective neuronal ablation in primary cell cultures.

(a) Rapid loss of action potential firing in DRG neurons from Avil-Cre::SNAPCaaX mice labelled with BG-Fluorescein upon illumination. Insets show action potential shape at beginning and end of trace. Scale bars 50μm (b-d) DRG from Avil-Cre::SNAPCaaX mice labelled with BG-Fluorescein and AnnexinV-Cy3. (b) Immediately after illumination. (c) 24 hours following illumination. (d) 24 hours without illumination. (e-f) DRG from Avil-Cre::SNAPCaaX mice labelled with negative control BG-505-Star and AnnexinV-Cy3. (e) Immediately after illumination. (f) 24 hours following illumination. (g) Quantification of AnnexinV-Cy3 staining. (h-k) Axonal degeneration and cell death in cultured DRG neurons from Avil-Cre::SNAPCaaX mice labelled with BG-fluorescein and AnnexinV-Cy3 and illuminated. (h) 1 hour after illumination. (i) 4 hours after illumination axonal degeneration is apparent. (j) 8 hours after illumination cell bodies show damaged morphology. (k) 16 hours after illumination prominent AnnexinV-Cy3 labelling is evident. (l-m) DRG/microglial co-cultures from Avil-Cre::SNAPCaaX mice labelled with BG-Fluorescein, AnnexinV-Cy3, and IB4. (l) Prior to illumination. (m) 24 hours after illumination. Scale bars 50μm.

Supplementary Figure 8 DNA fragmentation and apoptosis in DRG and skin sections.

(a) Lumbar DRG sections from Avil-Cre::SNAPCaaX mice injected with BG-Fluorescein into the hind paw, illuminated in vivo and subjected to the TUNEL assay 4 days after illumination. (b) DRG sections from whole mount Deleter-Cre::SNAPCaaX DRG labelled with BG-Fluorescein, illuminated ex vivo and subjected to the TUNEL assay. (c) Treatment of DRG sections with DNase I as an internal positive control for the TUNEL assay. Phase contrast images are shown below. (d) Skin sections from Avil-Cre::SNAPCaaX mice labelled with BG-Fluorescein, illuminated in vivo and subjected to the TUNEL assay 4 days after illumination. (e) Skin sections from Deleter-Cre::SNAPCaaX mice labelled with BG-Fluorescein, illuminated and subjected to the TUNEL assay. (f) Treatment of skin sections with DNase I as an internal positive control for the TUNEL assay. Scale bars 50μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 7061 kb)

In vivo labelling of hairy skin.

Volume rendering of hairy skin from Avil-Cre::SNAPCaaX mice stained with TMR-Star (white) and Hoechst 33342 (blue). (AVI 3296 kb)

In vivo labelling of glabrous skin.

Volume rendering of glabrous skin from Avil-Cre::SNAPCaaX mice stained with TMR-Star (red) and Hoechst 33342 (blue). (AVI 6156 kb)

Neuronal/microglial co-cultures before illumination.

Labelling of DRG/microglia co-cultures from Avil-Cre::SNAPCaaX mice with BG-Fluorescein (neurons, green), IB4 (microglia, cyan) and AnnexinV-Cy3 (red). Microglia can be seen scanning healthy neurons. (AVI 1088 kb)

Neuronal/microglial co-cultures after illumination.

DRG/microglia co-cultures from Avil-Cre::SNAPCaaX mice labelled with BG-Fluorescein (neurons, green), IB4 (microglia, cyan) and AnnexinV-Cy3 (red) 24 hours post illumination. Microglia are engulfing dead neurons and neuronal debris. (AVI 1195 kb)

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Yang, G., de Castro Reis, F., Sundukova, M. et al. Genetic targeting of chemical indicators in vivo. Nat Methods 12, 137–139 (2015). https://doi.org/10.1038/nmeth.3207

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