Production of new fluorescent proteins

Dr. Robert Campbell and his team, as the Optogenetic Protein Engineering Node of the Canadian Neurophotonics Platform, have been very active in the development of new fluorescent proteins that can be used as tools to for imaging dynamic biochemical events in live cells and tissues. The Campbell lab distribute these tools to cell biologists and neuroscientists who apply them to address questions ranging from fundamental mechanisms in cell biology, to the underlying causes of mental illness, to the development of novel therapeutics.

Lists of fluorescent proteins developed by Robert Campbell, Canadian Neurophotonics Platform Core member

• https://www.fpbase.org/collection/384/
• https://www.addgene.org/Robert_Campbell/

Development of improved genetically encoded contrast agents for photoacoustic microscopy (Robert E. Campbell’s team)

• Y. Li (equal contribution), A. Forbrich (equal contribution), J. Wu, P. Shao, R. E. Campbell* and R. Zemp*, “Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging”. Sci. Rep., 2016, 6, 22129.
• R.K.W. Chee, Y. Li, W. Zhang, R.E. Campbell, and R.J. Zemp*, “In vivo photoacoustic difference-spectra imaging of bacteria using photoswitchable chromoproteins”, J. Biomed. Opt., 2018, 23, 106006.

Development of a new series of genetically encoded voltage indicators for imaging of neuronal activity (Robert E. Campbell’s team)

• A.S. Abdelfattah, S.L. Farhi (equal contribution), Y. Zhao (equal contribution), D. Brinks, P. Zou, A. Ruangkittisakul, J. Platisa, V.A. Pieribone, K. Ballanyi, A.E. Cohen, and R.E. Campbell*, “A bright and fast red fluorescent protein voltage indicator that reports neuronal activity in organotypic brain slices”. J. Neurosci. 2016, 36, 2458–2472.

Invention of a new system of optogenetic control based on photcleavage of a genetically encoded protein (Robert E. Campbell’s team)

• W. Zhang (equal contribution), A.W. Lohman (equal contribution), Y. Zhuravlova, X. Lu, M.D. Wiens, H. Hoi, S. Yaganoglu, M.A. Mohr, E.N. Kitova, J.S. Klassen, P. Pantazis, R.J. Thompson, and R.E. Campbell*, “Optogenetic Control with a Photocleavable Protein, PhoCl”, Nat. Methods, 2017, 14, 391-394.

Development of  improved green, yellow, red, and near-infrared fluorescent genetically encoded Ca2+ indicators for imaging of neuronal activity (Robert E. Campbell’s team)

• Y. Qian (equal contribution), K.D. Piatkevich (equal contribution), B. McLarney (equal contribution), A.S. Abdelfattah, S. Mehta, M.H. Murdock, S. Gottschalk, R.S. Molina, W. Zhang, Y. Chen, J. Wu, M. Drobizhev, T.E. Hughes, J. Zhang, E.R. Schreiter, S. Shoham, D. Razansky, E.S. Boyden, and R.E. Campbell*, “A genetically encoded near-infrared fluorescent calcium ion indicator”, Nat. Methods, 2019, 16, 171–174.
• Y. Shen, H. Dana, A.S. Abdelfattah, R. Patel, J. Shea, R.S. Molina, B. Rawal, V. Rancic, Y.-F. Chang, L. Wu, Y. Chen, Y. Qian, M.D. Wiens, N. Hambleton, K. Ballanyi, T.E. Hughes, M. Drobizhev, D.S. Kim, M. Koyama, E.R. Schreiter, and R.E. Campbell*, “A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein eqFP578”, BMC Biol., 2018, 16, 9.
• Y. (Yufeng) Zhao, D. Bushey, Y. (Yongxin) Zhao, E.R. Schreiter, D.J. Harrison, A.M. Wong*, and R.E. Campbell*, “Inverse-response Ca2+ indicators for optogenetic visualization of inhibitory synapse activity”, Sci. Rep., 2018, 8, 11758.
• Landon Zarowny (equal contribution), Abhi Aggarwal (equal contribution), Virginia Rutten, Ilya Kolb, The GENIE Project, Ronak Patel, Hsin-Yi Huang, Yu-Fen Chang, Tiffany Phan, Richard Kanyo, Misha Ahrens, W. Ted Allison, Kaspar Podgorski, Robert E. Campbell*, “A bright and high-performance genetically encoded Ca2+ indicator based on mNeonGreen fluorescent protein”, Submitted. Posted on bioRxiv Jan. 17, 2020, doi: https://doi.org/10.1101/2020.01.16.909291

Development of a bright and high-performance bioluminescent Ca2+ indicator (Robert E. Campbell’s team)

• Y. Qian, V. Rancic, J. Wu, K. Ballanyi, and R.E. Campbell* , “A bioluminescent Ca2+ indicator based on a topological variant of GCaMP6s”, ChemBioChem, 2019, 20, 516-520.

Development of first-generation genetically encoded K+ sensors (Robert E. Campbell’s team)

• Y. Shen, S-Y. Wu, V. Rancic, A. Aggarwal, Y. Qian, S.-I. Miyashita, K. Ballanyi, R.E. Campbell*, and M. Dong*, “Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentrations”, Commun. Biol., 2019, 2, 18.

Development of red fluorescent glutamate indicators for visualization of excitatory neurotransmission (Robert E. Campbell’s team)

• J. Wu, A.S. Abdelfattah, H. Zhou, A. Ruangkittisakul, Y. Qian, K. Ballanyi and R.E. Campbell*, “Genetically Encoded Glutamate Indicators with Altered Color and Topology”, ACS Chem. Biol., 2018, 13, 1832–1837.

Developing use of optical approaches to study functional features of central synapses function and plasticity

The uOttawa node has developed experimental and computational expertise to meaningfully use optical approaches to study functional features of central synapses function and plasticity.  The group uses multiphoton imaging and uncaging of caged molecules to activate and monitor the electrical activity (Lee et al., Neuron 2016), the trafficking of receptors (George et al., eLife, 2015) and neurotransmitter release at single synapses (Soares et al., 2017).

The group recently developed experimental and modern computational approaches to calibrate the properties of the glutamate optical reporter iGluSNFR and established its potential for the characterization of synaptic properties. This combined optical, electrophysiological and computational approach showed that this sensor is a reliable and linear encoder of glutamate concentration at individual synapses, with sufficient potency to detect single endogenous vesicle release events. A model-based statistical toolbox was developed to extract physiological properties (vesicle number, size and release probability) from the optical signal (Soares et al. 2019).

The group is also using modern computational approaches to model synaptic, cellular, network and behavioural dynamics from electrophysiological and optogenetic approaches.

Viral Vector Production Core Growth

The fluorescent proteins created by the Optogenetic Protein Engineering Node of the Canadian Neurophotonics Platform are produced by the viral vector production core, to be tested by the testing nodes of the CNP.  These tools are also shared with the scientific community.

The Viral Vector production core has grown significantly over the last years, both in terms of number of viral vectors distributed, and in the number of clients.

The viral vector core has also developed and implemented new approaches and systems to improve services, including

• Rabies viruses (self inactivating and G-deleted, G or EnvA peudotyped)
• New capsids for systemic injection
• Purification from culture medium
• qPCR Titration protocols optimization and ddPCR implementation
• Development of serotype testing kits
• EM characterization of AAV particles
• Online ordering via tools.neurophotonics.ca

 

Other technologies developed by the Neurophotonics Platform include

• Wireless technologies with Benoît Gosselin and Doric Lenses
• Axicon-based volumetric microscopy (patent obtained by Yves De Koninck’s team to  protect intellectual property)
• SLAM superresolution microscopy (patent obtained by Yves De Koninck’s team to  protect intellectual property)
• Development & improvements of new epidural probes (in conjunction with optogenetics)