A Guide to Creating and Testing New INTRSECT Constructs

Lief E. Fenno1, Joanna Mattis2, Charu Ramakrishnan3, Karl Deisseroth4

1 Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California, 2 Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania, 3 Department of Bioengineering, Stanford University, Stanford, California, 4 Howard Hughes Medical Institute, Stanford University, Stanford, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.39
DOI:  10.1002/cpns.30
Online Posting Date:  July, 2017
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Abstract

As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single‐recombinase‐dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase‐dependent viruses with recombinase‐expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features. This method includes the design and utilization of both viruses and transgenic animals: these tools are inherently flexible and modular and may be used in different combinations to achieve the desired gene expression pattern. © 2017 by John Wiley & Sons, Inc.

Keywords: INTRSECT; neuroscience; optogenetics; synthetic biology; virus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: INTRSECT Targeting of Genetic Marker X and Genetic Marker Y with a Fusion Gene
  • Alternate Protocol 1: INTRSECT Targeting of Genetic Marker X and Downstream Projection Y
  • Alternate Protocol 2: INTRSECT Targeting of Genetic Marker X and Not Genetic Marker Y with a Fusion Gene
  • Alternate Protocol 3: INTRSECT Targeting of Genetic Markers with a Non‐Fusion Gene
  • Support Protocol 1: Immunohistochemistry
  • Support Protocol 2: INTRSECT Construct Analysis Using RT‐PCR
  • Support Protocol 3: INTRSECT Construct Analysis Using Flow Cytometry
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: INTRSECT Targeting of Genetic Marker X and Genetic Marker Y with a Fusion Gene

  Materials
  • Molecular cloning software: options include commercial (VectorNTI, SnapGene), free (ApE), and cloud‐based (http://www.everyvector.com) software
  • Full sequence of gene of interest
  • INTRSECT backbone vector and sequence (available from http://www.optogenetics.org)
  • Recombinase expression constructs
  • E. coli suitable for cloning constructs with repetitive sequence (Stbl3): the AAV backbone used for cloning INTRSECT constructs and making AAV has long, repetitive sequences that may impair construct stability in bacteria able to homologously recombine DNA
  • Endotoxin‐free large‐scale DNA preparation kit; endotoxin‐free DNA is important for primary neuron transfection and virus production
  • Cultured mammalian cell line of interest (HEK‐293, primary neuronal cultures, etc.)
  • Transfection reagents (Lipofectamine, CaPO 4, etc.).
  • Cre‐conditional (cDIO) and Flp‐conditional (fDIO) adeno‐associated viruses: many options are available at http://www.optogenetics.org
  • Cre;Flp double transgenic driver mice: mice may be acquired from many sources, including the Jackson Laboratory (http://www.jax.org), the GENSAT project (http://www.gensat.org), and the Allen Institute (http://www.brain‐map.org)
  • Wild‐type (WT) mice as controls
  • Epifluorescence microscope
  • Additional reagents and equipment for molecular cloning techniques (Ausubel et al., ) and stereotactic injection of adenoviral vectors (unit 4.24; Puntel et al., )

Alternate Protocol 1: INTRSECT Targeting of Genetic Marker X and Downstream Projection Y

  Additional Materials (also see protocol 1Basic Protocol)
  • Cre transgenic driver mice: mice may be acquired from many sources, including the Jackson Labs (http://www.jax.org), the GENSAT project (http://www.gensat.org), or the Allen Institute (http://www.brain‐map.org)
  • Flp‐expressing retrograde virus: obtain a retrograde virus [e.g., LT‐HSV (Kim et al., )] encoding Flp recombinase along with a fluorophore to enable easy identification of expressing cells; as an alternative to creating a fusion protein (e.g., Flp‐mCherry), it may be preferable to separate the fluorophore from the recombinase using an IRES sequence (e.g., mCherry‐IRES‐Flp or mCherry‐p2a‐Flp), resulting in separate translation of the two proteins and possibly leading to higher activity levels of the recombinase
  • Cre‐conditional (cDIO) adeno‐associated viruses (many options are available at http://www.optogenetics.org)
  • Cre‐ and Flp‐conditional (C on/F on) INTRSECT adeno‐associated virus (AAV) encoding gene of interest (see protocol 1Basic Protocol)

Alternate Protocol 2: INTRSECT Targeting of Genetic Marker X and Not Genetic Marker Y with a Fusion Gene

  Materials
  • Mouse injected with recombinant AAV vector ( protocol 1Basic Protocol or protocol 2 or 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% (w/v) paraformaldehyde (PFA)
  • 30% (w/v) sucrose in PBS (gentle heating and stirring are required to dissolve sucrose; do not boil, filter sterilize through 0.2‐µm filter)
  • Cryoprotectant (25% glycerol, 30% ethylene glycol, 45% PBS, pH 6.7; filter sterilize 0.2 µm)
  • Triton X‐100 (Sigma)
  • Normal donkey serum (NDS; Jackson ImmunoResearch, cat. no. 017‐000‐121)
  • Primary antibody (Jackson ImmunoResearch)
  • Fluorophore‐labeled secondary antibody (Jackson ImmunoResearch)
  • DAPI (optional)
  • PVA‐DABCO (Sigma, cat. no. 10981)
  • Freezing microtome (Leica SM2000 R)
  • Slides (Superfrost Plus recommended; VWR, cat. no. 48311‐703) and coverslips (VWR, cat. no. 48393 059)
  • Additional reagents and equipment for immunohistochemical techniques (unit 1.1; Gerfen, )

Alternate Protocol 3: INTRSECT Targeting of Genetic Markers with a Non‐Fusion Gene

  Materials
  • Cultured cell line (we use HEK cells; ATCC #CRL‐1573 or #CRL‐3216; ThermoFisher, cat. no. R70007)
  • INTRSECT construct (http://www.optogenetics.org)
  • Recombinase expression constructs (http://www.optogenetics.org)
  • Transfection reagents (Lipofectamine; ThermoFisher, cat. no. 11668030)
  • mRNA isolation and RT‐PCR reagents (SuperScript III One‐Step; ThermoFisher, cat. no. 12574018)
  • Direction‐specific primers
  • Standard PCR reagents (Platinum Taq DNA Polymerase; ThermoFisher, cat. no. 10966018)
  • Gel purification reagents (optional; Qiagen, cat. no. 28704)
  • Sequencing primers (optional)
  • 12‐well culture plates
  • Additional reagents and equipment for molecular cloning techniques (Ausubel et al., )

Support Protocol 1: Immunohistochemistry

  Materials
  • Cultured cell line (we use HEK cells; ATCC #CRL‐1573 or #CRL‐3216; ThermoFisher, cat. no. R70007)
  • INTRSECT construct (http://www.optogenetics.org)
  • Recombinase expression constructs (http://www.optogenetics.org)
  • Transfection reagents (Lipofectamine; ThermoFisher, cat. no. 11668030)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 mg/ml (1000×) propidium iodide or other spectrally appropriate vital dye
  • 0.25% trypsin (Gibco, cat. no. 12604013)
  • 24‐well culture plates
  • Flow cytometer–compatible tubes
  • 95°C heat block
  • Flow cytometer
  • Additional reagents and equipment for molecular cloning techniques (Ausubel et al., )
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Figures

Videos

Literature Cited

 
  Atasoy, D., Aponte, Y., Su, H. H., & Sternson, S. M. (2008). A FLEX switch targets Channelrhodopsin‐2 to multiple cell types for imaging and long‐range circuit mapping. The Journal of Neuroscience, 28, 7025–7030. doi: 10.1523/JNEUROSCI.1954‐08.2008.
  Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., … Struhl, K. (Eds.). (2017). Current Protocols in Molecular Biology. Hoboken, NJ: John Wiley & Sons.
  Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth, K. (2005). Millisecond‐timescale, genetically targeted optical control of neural activity. Nature Neuroscience, 8, 1263–1268. doi: 10.1038/nn1525.
  Bru, T., Salinas, S., & Kremer, E. J. (2010). An update on canine adenovirus type 2 and its vectors. Viruses, 2, 2134–2153. doi: 10.3390/v2092134.
  Brunak, S., Engelbrecht, J., & Knudsen, S. (1991). Prediction of human mRNA donor and acceptor sites from the DNA sequence. Journal of Molecular Biology, 220, 49–65. doi: 10.1016/0022‐2836(91)90380‐O.
  Buchholz, F., Angrand, P. O., & Stewart, A. F. (1998). Improved properties of FLP recombinase evolved by cycling mutagenesis. Nature Biotechnology, 16, 657–662. doi: 10.1038/nbt0798‐657.
  Chapman, B. S., Thayer, R. M., Vincent, K. A., & Haigwood, N. L. (1991). Effect of intron A from human cytomegalovirus (Towne) immediate‐early gene on heterologous expression in mammalian cells. Nucleic Acids Research, 19, 3979–3986. doi: 10.1093/nar/19.14.3979.
  Chung, K., Wallace, J., Kim, S. Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., … Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems. Nature, 497, 332–337. doi: 10.1038/nature12107.
  Crick, F. H. (1979). Thinking about the brain. Scientific American, 241, 219–232. doi: 10.1038/scientificamerican0979‐219.
  Fenno, L. E., Mattis, J., Ramakrishnan, C., Hyun, M., Lee, S. Y., He, M., … Deisseroth, K. (2014). Targeting cells with single vectors using multiple‐feature Boolean logic. Nature Methods, 11, 763–772. doi: 10.1038/nmeth.2996.
  Fenno, L., Yizhar, O., & Deisseroth, K. (2011). The development and application of optogenetics. Annual Review of Neuroscience, 34, 389–412. doi: 10.1146/annurev‐neuro‐061010‐113817.
  Gao, K., Masuda, A., Matsuura, T., & Ohno, K. (2008). Human branch point consensus sequence is yUnAy. Nucleic Acids Research, 36, 2257–2267. doi: 10.1093/nar/gkn073.
  Gerfen, C. R. (2003). Basic neuroanatomical methods. Current Protocols in Neuroscience, 00, 1.1.1–1.1.11. doi: 10.1002/0471142301.ns0101s23.
  Gradinaru, V., Zhang, F., Ramakrishnan, C., Mattis, J., Prakash, R., Diester, I., … Deisseroth, K. (2010). Molecular and cellular approaches for diversifying and extending optogenetics. Cell, 141, 154–165. doi: 10.1016/j.cell.2010.02.037.
  Grieger, J. C., Choi, V. W., & Samulski, R. J. (2006). Production and characterization of adeno‐associated viral vectors. Nature Protocols, 1, 1412–1428. doi: 10.1038/nprot.2006.207.
  Grimm, D., Lee, J. S., Wang, L., Desai, T., Akache, B., Storm, T. A., & Kay, M. A. (2008). In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno‐associated viruses. Journal of Virology, 82, 5887–5911. doi: 10.1128/JVI.00254‐08.
  Ishida, N., Ueda, S., Hayashida, H., Miyata, T., & Honjo, T. (1982). The nucleotide sequence of the mouse immunoglobulin epsilon gene: Comparison with the human epsilon gene sequence. The EMBO Journal, 1, 1117–1123.
  Kato, S., Kobayashi, K., Inoue, K., Kuramochi, M., Okada, T., Yaginuma, H., … Kobayashi, K. (2011a). A lentiviral strategy for highly efficient retrograde gene transfer by pseudotyping with fusion envelope glycoprotein. Human Gene Therapy, 22, 197–206. doi: 10.1089/hum.2009.179.
  Kato, S., Kuramochi, M., Takasumi, K., Kobayashi, K., Inoue, K., Takahara, D., … Kobayashi, K. (2011b). Neuron‐specific gene transfer through retrograde transport of lentiviral vector pseudotyped with a novel type of fusion envelope glycoprotein. Human Gene Therapy, 22, 1511–1523. doi: 10.1089/hum.2011.111.
  Kim, E. J., Jacobs, M. W., Ito‐Cole, T., & Callaway, E. M. (2016). Improved monosynaptic neural circuit tracing using engineered rabies virus glycoproteins. Cell Reports. doi: 10.1016/j.celrep.2016.03.067.
  Kim, S. Y., Adhikari, A., Lee, S. Y., Marshel, J. H., Kim, C. K., Mallory, C. S., … Deisseroth, K. (2013). Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature, 496, 219–223. doi: 10.1038/nature12018.
  Lammel, S., Lim, B. K., & Malenka, R. C. (2013). Reward and aversion in a heterogeneous midbrain dopamine system. Neuropharmacology, 76, 351–359. doi: 10.1016/j.neuropharm.2013.03.019.
  Luo, L., Callaway, E. M., & Svoboda, K. (2008). Genetic dissection of neural circuits. Neuron, 57, 634–660. doi: 10.1016/j.neuron.2008.01.002.
  Marcinkiewcz, C. A., Mazzone, C. M., D'Agostino, G., Halladay, L. R., Hardaway, J. A., DiBerto, J. F., … Kash, T. L. (2016). Serotonin engages an anxiety and fear‐promoting circuit in the extended amygdala. Nature, 537, 97–101. doi: 10.1038/nature19318.
  Marshel, J. H., Mori, T., Nielsen, K. J., & Callaway, E. M. (2010). Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron, 67, 562–574. doi: 10.1016/j.neuron.2010.08.001.
  McLeod, M., Craft, S., & Broach, J. R. (1986). Identification of the crossover site during FLP‐mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Molecular and Cellular Biology, 6, 3357–3367. doi: 10.1128/MCB.6.10.3357.
  Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Research, 10, 459–472. doi: 10.1093/nar/10.2.459.
  Osakada, F., Mori, T., Cetin, A. H., Marshel, J. H., Virgen, B., & Callaway, E. M. (2011). New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron, 71, 617–631. doi: 10.1016/j.neuron.2011.07.005.
  Oyibo, H. K., Znamenskiy, P., Oviedo, H. V., Enquist, L. W., & Zador, A. M. (2014). Long‐term Cre‐mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus. Frontiers in Neuroanatomy, 8, 86. doi: 10.3389/fnana.2014.00086.
  Puntel, M., Kroeger, K. M., Sanderson, N. S., Thomas, C. E., Castro, M. G., & Lowenstein, P. R. (2010). Gene transfer into rat brain using adenoviral vectors. Current Protocols in Neuroscience, 50, 4.24.1–4.24.49. doi: 10.1002/0471142301.ns0424s50.
  Raymond, C. S., & Soriano, P. (2007). High‐efficiency FLP and PhiC31 site‐specific recombination in mammalian cells. PloS One, 2, e162. doi: 10.1371/journal.pone.0000162.
  Reardon, T. R., Murray, A. J., Turi, G. F., Wirblich, C., Croce, K. R., Schnell, M. J., … Losonczy, A. (2016). Rabies virus CVS‐N2c(DeltaG) strain enhances retrograde synaptic transfer and neuronal viability. Neuron, 89, 711–724. doi: 10.1016/j.neuron.2016.01.004.
  Sauer, B., & McDermott, J. (2004). DNA recombination with a heterospecific Cre homolog identified from comparison of the pac‐c1 regions of P1‐related phages. Nucleic Acids Research, 32, 6086–6095. doi: 10.1093/nar/gkh941.
  Saunders, A., Johnson, C. A., & Sabatini, B. L. (2012). Novel recombinant adeno‐associated viruses for Cre activated and inactivated transgene expression in neurons. Frontiers in Neural Circuits, 6, 47. doi: 10.3389/fncir.2012.00047.
  Schlake, T., & Bode, J. (1994). Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry, 33, 12746–12751. doi: 10.1021/bi00209a003.
  Stamatakis, A. M., Jennings, J. H., Ung, R. L., Blair, G. A., Weinberg, R. J., Neve, R. L., … Stuber, G. D. (2013). A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward. Neuron, 80, 1039–1053. doi: 10.1016/j.neuron.2013.08.023.
  Sternberg, N., Hamilton, D., Austin, S., Yarmolinsky, M., & Hoess, R. (1981). Site‐specific recombination and its role in the life cycle of bacteriophage P1. Cold Spring Harbor Symposia on Quantitative Biology 45(Pt 1), 297–309. doi: 10.1101/SQB.1981.045.01.042.
  Suzuki, E., & Nakayama, M. (2011). VCre/VloxP and SCre/SloxP: New site‐specific recombination systems for genome engineering. Nucleic Acids Research, 39, e49. doi: 10.1093/nar/gkq1280.
  Tovote, P., Esposito, M. S., Botta, P., Chaudun, F., Fadok, J. P., Markovic, M., … Luthi, A. (2016). Midbrain circuits for defensive behaviour. Nature, 534, 206–212. doi: 10.1038/nature17996.
  Tsai, H. C., Zhang, F., Adamantidis, A., Stuber, G. D., Bonci, A., de Lecea, L., & Deisseroth, K. (2009). Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science, 324, 1080–1084. doi: 10.1126/science.1168878.
  Van Bockstaele, E. J., & Pickel, V. M. (1995). GABA‐containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain. Brain Research, 682, 215–221. doi: 10.1016/0006‐8993(95)00334‐M.
  Wickersham, I. R., Lyon, D. C., Barnard, R. J., Mori, T., Finke, S., Conzelmann, K. K., … Callaway, E. M. (2007). Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron, 53, 639–647. doi: 10.1016/j.neuron.2007.01.033.
  Witten, I. B., Steinberg, E. E., Lee, S. Y., Davidson, T. J., Zalocusky, K. A., Brodsky, M., … Deisseroth, K. (2011). Recombinase‐driver rat lines: Tools, techniques, and optogenetic application to dopamine‐mediated reinforcement. Neuron, 72, 721–733. doi: 10.1016/j.neuron.2011.10.028.
  Xia, Y., Driscoll, J. R., Wilbrecht, L., Margolis, E. B., Fields, H. L., & Hjelmstad, G. O. (2011). Nucleus accumbens medium spiny neurons target non‐dopaminergic neurons in the ventral tegmental area. The Journal of Neuroscience, 31, 7811–7816. doi: 10.1523/JNEUROSCI.1504‐11.2011.
  Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., & Deisseroth, K. (2011). Optogenetics in neural systems. Neuron, 71, 9–34. doi: 10.1016/j.neuron.2011.06.004.
  Zhang, M. Q. (1998). Statistical features of human exons and their flanking regions. Human Molecular Genetics, 7, 919–932. doi: 10.1093/hmg/7.5.919.
Internet Resources
  http://www.optogenetics.org
  Optogenetics Resource Center: INTRSECT constructs and viruses.
  http://www.addgene.org
  Addgene: Viral core facilities.
  http://www.med.unc.edu/genetherapy/vectorcore
  UNC Vector Core.
  http://med.stanford.edu/gvvc/
  Stanford Gene Vector and Virus Core.
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