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

  • 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

  • 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

  • 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

  • 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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Internet Resources
  Optogenetics Resource Center: INTRSECT constructs and viruses.
  Addgene: Viral core facilities.
  UNC Vector Core.
  Stanford Gene Vector and Virus Core.
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