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Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling

Graybuck, Lucas T. and Daigle, Tanya and Sedeño-Cortés, Adriana and Walker, Miranda and Kalmbach, Brian and Lenz, Garreck H. and Nguyen, Thuc Nghi and Garren, Emma and Kim, Tae Kyung and Sieverts, La'Akea and Bendrick, Jacqueline L. and Zhou, Thomas and Mortrud, Marty and Yao, Shenqin and Cetin, Ali H. and Larsen, Rachael and Esposito, Luke and Gore, Bryan and Szelenyi, Eric R. and Morin, Elyse and Mich, John K. and Dee, Nick and Goldy, Jeff and Smith, Kimberly A.. and Yao, Zizhen and Gradinaru, Viviana and Sunkin, Susan M. and Lein, Ed and Levi, Boaz P. and Ting, Jonathan and Zeng, Hongkui and Tasic, Bosiljka (2020) Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling. . (Unpublished)

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The rapid pace of cell type identification by new single-cell analysis methods has not been met with efficient experimental access to the newly discovered types. To enable flexible and efficient access to specific neural populations in the mouse cortex, we collected chromatin accessibility data from individual cells and clustered the single-cell data to identify enhancers specific for cell classes and subclasses. When cloned into adeno-associated viruses (AAVs) and delivered to the brain by retro-orbital injections, these enhancers drive transgene expression in specific cell subclasses in the cortex. We characterize several enhancer viruses in detail to show that they result in labeling of different projection neuron subclasses in mouse cortex, and that one of them can be used to label the homologous projection neuron subclass in human cortical slices. To enable the combinatorial labeling of more than one cell type by enhancer viruses, we developed a three-color Cre-, Flp- and Nigri- recombinase dependent reporter mouse line, Ai213. The delivery of three enhancer viruses driving these recombinases via a single retroorbital injection into a single Ai213 transgenic mouse results in labeling of three different neuronal classes/subclasses in the same brain tissue. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Graybuck, Lucas T.0000-0002-8814-6818
Daigle, Tanya0000-0001-9700-8452
Sedeño-Cortés, Adriana0000-0001-7959-8970
Walker, Miranda0000-0002-3767-1591
Kalmbach, Brian0000-0003-3136-8097
Lenz, Garreck H.0000-0002-3233-0763
Nguyen, Thuc Nghi0000-0002-6466-5883
Kim, Tae Kyung0000-0001-9646-5969
Sieverts, La'Akea0000-0002-1385-086X
Bendrick, Jacqueline L.0000-0002-2694-9914
Yao, Shenqin0000-0003-2992-4752
Cetin, Ali H.0000-0003-1510-0517
Morin, Elyse0000-0002-7310-1561
Mich, John K.0000-0002-1626-1139
Dee, Nick0000-0002-2831-9254
Goldy, Jeff0000-0001-5140-6922
Smith, Kimberly A..0000-0002-3142-1970
Yao, Zizhen0000-0002-9361-5607
Gradinaru, Viviana0000-0001-5868-348X
Sunkin, Susan M.0000-0001-9893-3834
Lein, Ed0000-0001-9012-6552
Levi, Boaz P.0000-0002-8346-872X
Zeng, Hongkui0000-0002-0326-5878
Tasic, Bosiljka0000-0002-6861-4506
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Posted April 21, 2020. We could not have performed this study without the support of the Allen Institute Animal Care Team for mouse husbandry, the Allen Institute Transgenic Colony Management team for colony management, the Allen Institute Laboratory Animal Services team for preparation and delivery of experimental animals; Tamara Caspar, Kirsten Chrichton, Matthew Kroll, Josef Sulc, and Herman Tung for tissue processing; Nadiya Shapovalova, Daniel Hirschstein, and Susan Bort for FACS; Natalie Weed and Victoria Omstead for retro-orbital injections; Refugio Martinez and Ximena Opitz-Araya for cloning; and Darren Bertagnolli, Michael Tieu, Delissa McMillen, Thanh Pham, Christine Rimorin, Katelyn Ward, Alexandra Glandon, and Amy Torkelson for scRNA-seq processing. The authors also thank Andrew Hill and Darren Cusanovich for assistance in accessing and reusing data published in Cusanovich and Hill et al. (2018). We thank Advanced Cell Diagnostics for validation of the Fam84b probe and for early access to the RNAscope HiPlex assays. The project described was supported by award number 1R01DA036909-01 REVISED from the National Institute on Drug Abuse to B.T. and H.Z., and by NIH BRAIN Initiative awards 1RF1MH121274-01 to B.T., T.L.D. and H.Z., and 1RF1MH114126-01 to B.P.L, J.T., E.L, and B.T. from the National Institute of Mental Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health, the National Institute on Drug Abuse, or the National Institute of Mental Health. The authors thank the Allen Institute founder, Paul G. Allen, for his vision, encouragement, and support. Author contributions: B.T., L.T.G., and T.L.D. designed the study; J.T.T., B.P.L, and J.K.M. provided viral genome constructs and cloning protocols; G.H.L. and M.W. cloned enhancers and generated viral constructs. T.L.D., L.S., and G.H.L., M.W. designed and generated the Ai213 transgenic mouse line. J.T.T. identified the mscRE4 enhancer core and performed cloning experiments for the concatemer and provided the hI56i-iCre-4X2C virus. A.S.-C., T.N., and L.T.G. performed scATAC-seq experiments. B.K. and J.T. performed electrophysiology experiments. S.Y., M.M., and T.Z. performed viral packaging and purification. T.N., T.K., and M.M. performed retrograde injections. M.W. and L.S. performed retro-orbital injections. T.N., and B.G. E.S. performed ICV and stereotaxic injections. M.W., J.B., E.M., L.S. and T.L.D. performed IHC experiments, imaging, cell counting and analysis. T.N. and E.G. performed mFISH validation experiments. N.D. managed tissue processing for RNA-seq experiments. K.S. managed RNA-seq experiments. R.L. and L.E. managed the transgenic line colonies. A.H.C. managed virus production. Z.Y. and L.T.G. performed RNA-seq analysis. L.T.G. and A.S.-C. performed scATAC-seq analysis. B.K. performed electrophysiology data analysis. S.M.S. provided project management. H.Z. and E.L. lead the Cell Types Program at the Allen Institute. L.T.G., T.L.D., and B.T. wrote the manuscript, with input from all coauthors. Competing interests: L.T.G., T.L.D., J.T.T., J.K.M., B.P.L., E.L., B.K., H.Z., and B.T. are inventors on several U.S. provisional patent applications related to this work. All authors declare no other competing interests. Data and materials availability: scATAC-seq and scRNA-seq data will be deposited to GEO. Software code used for data analysis and visualization is available from GitHub at An R package for analysis of low-coverage accessibility and transcriptomics (lowcat) is available on GitHub at lowcat/, and an R package for generating figures based on the Allen Institute Common Coordinate Framework (cocoframer) is available at
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Record Number:CaltechAUTHORS:20200421-092814882
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Official Citation:Enhancer viruses and a transgenic platform for combinatorial cell subclass-specific labeling. Lucas T Graybuck, Tanya Daigle, Adriana Sedeño-Cortés, Miranda Walker, Brian Kalmbach, Garreck H Lenz, Thuc Nghi Nguyen, Emma Garren, Tae Kyung Kim, La'Akea Sieverts, Jacqueline L Bendrick, Thomas Zhou, Marty Mortrud, Shenqin Yao, Ali H Cetin, Rachael Larsen, Luke Esposito, Bryan Gore, Eric R Szelenyi, Elyse Morin, John K Mich, Nick Dee, Jeff Goldy, Kimberly A Smith, Zizhen Yao, Viviana Gradinaru, Susan M Sunkin, Ed Lein, Boaz P Levi, Jonathan Ting, Hongkui Zeng, Bosiljka Tasic. bioRxiv 525014; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102693
Deposited By: Tony Diaz
Deposited On:21 Apr 2020 16:39
Last Modified:21 Apr 2020 16:39

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