Manipulating Gene Expression in Projection‐Specific Neuronal Populations Using Combinatorial Viral Approaches

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The mammalian brain contains tremendous structural and genetic complexity that is vital for its function. The elucidation of gene expression profiles in the brain, coupled with the development of large?scale connectivity maps and emerging viral vector?based approaches for target?selective gene manipulation, now allows for detailed dissection of gene?circuit interfaces. This protocol details how to perform combinatorial viral injections to manipulate gene expression in subsets of neurons interconnecting two brain regions. This method uses stereotaxic injection of a retrograde transducing CAV2?Cre virus into one brain region, combined with injection of a locally transducing Cre?dependent AAV virus into another brain region. This technique is widely applicable to the genetic dissection of neural circuitry, as it enables selective expression of candidate genes, dominant?negatives, fluorescent reporters, or genetic tools within heterogeneous populations of neurons, based upon their projection targets. Curr. Protoc. Neurosci . 65:4.35.1?4.35.20. © 2013 by John Wiley & Sons, Inc.

Keywords: AAV; CAV; viral vector; neural circuit; stereotaxic surgery

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Introduction
Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces
Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain
Support Protocol 2: Adeno‐Associated Virus Production
Support Protocol 3: Production of CAV2‐Cre
Support Protocol 4: Brain Sectioning
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces

Materials

4% (v/v) Fluorogold (Fluorochrome)/0.9% (w/v) saline, or other appropriate dye
Wild‐type mouse, fluorescent reporter mouse (e.g., GFP or tdTomato at the Rosa26 genome locus), or Cre‐driver mouse (e.g., Jackson Laboratories or GENSAT), age and gender matched for targeting and experimental mice
Cre‐conditional or nonconditional adeno‐associated virus encoding reporter or gene of interest (AAV; 1 × 10⁹ particles/µl suggested; protocol 3 )
4% (w/v) paraformaldehyde (PFA), pH 7.4 (see recipe )
Canine associated virus‐2 encoding Cre recombinase (CAV2‐Cre; protocol 4 )
Mouse brain atlas (e.g., Paxinos and Franklin, ; Dong, ; http://www.brain‐map.org)

Additional reagents and equipment for carrying out stereotaxic injection ( protocol 2 ), perfusion fixation (unit 1.1 ), AAV virus production ( protocol 3 ), CAV2‐Cre virus production ( protocol 4 ), and tissue sectioning ( protocol 5 ).

Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain

Materials

Anesthesia (e.g., isoflurane)
Wild‐type mouse, fluorescent reporter mouse, or Cre‐driver mouse (e.g., Jackson Laboratories or GENSAT)
Topical anesthetic (e.g., 5% lidocaine cream)
Sterile water
Betadine
0.9% (w/v) saline, sterile
Virus, dye, or tracer of interest
Sutures or surgical adhesive

Stereotaxic equipment (e.g., Kopf)
- Sterotaxic frame
- 40× microscope attachment for stereotaxic frame
- Stereotaxic alignment indicator
- Stereotaxic drill
- Syringe holder
Electric razor
Sterile cotton swabs
Surgical tools:
- Scalpel
- Skin clamps
- Forceps
5‐µl injection syringe (e.g., Hamilton)
Microsyringe pump (e.g., World Precision Instruments)

Support Protocol 2: Adeno‐Associated Virus Production

Materials

∼5 × 10⁶ HEK293T/17 cells, low‐passage frozen stock (American Type Culture Collection)
HEK culture media (see recipe )
0.25% (w/v) trypsin‐EDTA (Life Technologies)
2M CaCl₂ , sterile
AAV shuttle vector with desired construct
pDG1 packaging vector
HEPES solution (see recipe )
Phosphate mix (see recipe )
Serum‐free HEK medium (see recipe )
Dry ice/95% (v/v) ethanol bath
40% (w/v) sucrose in phosphate‐buffered saline (PBS; appendix 2A ), sterile
1.37 g/ml CsCl solution (see recipe for CsCl solutions)
6× DNA loading buffer ( appendix 2A )
1% (w/v) agarose gel
0.5 µg/ml ethidium bromide
1× Hanks’ balanced salt solution (HBSS; appendix 2A )

Biosafety cabinet
Cell culture incubator
10‐ and 15‐cm tissue culture dishes
Hemacytometer
50‐ and 15‐ml conical centrifuge tubes
Pasteur pipets, sterile
37°C water bath
Clinical centrifuge
25 × 89–mm ultracentrifuge tubes (e.g., Beckman)
Ultracentrifuge (e.g., Beckman with SW‐27 and VTi‐65 rotors), or equivalent
13 × 51–mm heat‐seal ultracentrifuge tubes and sealer (e.g., Beckman)
1.5‐ml microcentrifuge tubes
0.6‐ml thin‐walled microcentrifuge tubes (e.g., Thermo Scientific)
2‐ to 3‐liter container
5‐ml syringe with 20‐G needle
10,000 molecular weight cutoff dialysis cassettes, 0.5‐ to 3‐ml capacity (e.g., Pierce)
0.2‐µm pore syringe filter
Parafilm

Additional reagents and equipment for carrying out agarose gel electrophoresis ( appendix 1N )

Support Protocol 3: Production of CAV2‐Cre

Materials

∼5 × 10⁶ DK‐ZEO cells, frozen stock (available from the authors upon request)
DK‐ZEO culture medium (see recipe )
0.25% (w/v) trypsin‐EDTA (Life Technologies)
CAV2‐Cre viral vector stock (available from the authors upon request)
Dry ice/95% (v/v) ethanol bath
CsCl solutions (see recipe )
Phosphate‐buffered saline (PBS), with CaCl₂ and MgCl₂ (see recipe )
Viral lysis solution: 0.1% (w/v) SDS/10 mM Tris·Cl, pH 7.4 (see appendix 2A )

Biosafety cabinet cell culture incubator
10‐ and 15‐cm tissue culture dishes
Cell scraper
50‐ml conical centrifuge tubes
Clinical centrifuge
25 × 89–mm and 14 × 89–mm ultracentrifuge tubes (e.g., Beckman)
Pasteur pipets
20‐G needle and 5‐ml syringe
Ultracentrifuge (e.g., Beckman with SW‐27 and SW‐41 rotors), or equivalent
PD‐10 columns (e.g., GE Life Sciences)
1.5‐ml microcentrifuge tubes
37°C and 56°C water baths
Spectrophotometer

Support Protocol 4: Brain Sectioning

Materials

Perfused mouse (e.g., see Basic Protocol)
4% (w/v) paraformaldehyde (PFA), pH 7.4 (see recipe )
30% (w/v) sucrose in PBS ( appendix 2A )
Freezing medium (e.g., O.C.T., TissueTek)
Dry ice/95% (v/v) ethanol bath
PBS ( appendix 2A )
Mounting medium (e.g., Fluoromount, Sigma‐Aldrich)

Small glass or plastic jar with a secure lid
Test tube rotator or rocker
Disposable embedding molds (Polysciences)
Rounded spatula or forceps
Cryostat
24‐ or 48‐well plate
Paintbrush
Glass slides

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Figures

Figure 4.35.1 Combinatorial viral delivery to study pathway‐specific gene function. (A ) Combined viral injections of a conditional AAV‐FLEX construct (red) into a region containing a neuronal population of interest and a CAV2‐Cre viral vector (blue) into a projection target of the neurons of interest. (B ) Local (red) AAV‐FLEX viral vector transduction and retrograde (blue) CAV2‐Cre transduction. (C ) Combinatorial viral‐mediated gene expression. Injection of CAV2‐Cre into a target region of interest (blue) of a Cre‐dependent reporter line (Ai14) illustrates the coverage area of viral vector injection and the number of neurons retrogradely transduced. Injection of AAV‐FLEX containing a conditional expression cassette of interest locally transduces neurons at the sight of injection; however, only neurons with combined CAV2‐Cre and AAV‐FLEX will express the transgene (red). For the color version of this figure, go to http://www.currentprotocols.com.

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Figure 4.35.2 Stereotaxic targeting of the y coordinate can be improved using a bregma‐lambda correction factor. Following incision, the scalp is held open with skin clamps, allowing access to the top of the skull. Bregma can be seen as the intersection of the midline rostral‐caudal fissure and the second major medial‐lateral fissure. Lambda can be identified as the most rostral medial‐lateral fissure. Stereotaxic x and y coordinates are set to zero at bregma. Moving from midline to the left is in the negative x direction and positive from midline to the right. Similarly, y coordinates are negative caudal to bregma and positive rostral to bregma. The bregma‐lambda correction factor ( F ) is the distance between bregma and lambda divided by 4.21 (the distance from bregma to lambda in a commonly used mouse brain atlas).

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Internet Resources
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	The AIBS Connectivity Atlas maps all the interconnections within the mouse brain.

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Manipulating Gene Expression in Projection‐Specific Neuronal Populations Using Combinatorial Viral Approaches

Abstract

Table of Contents

Materials

Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces

Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain

Support Protocol 2: Adeno‐Associated Virus Production

Support Protocol 3: Production of CAV2‐Cre

Support Protocol 4: Brain Sectioning

Figures

Videos

Literature Cited