Manipulating Gene Expression in Projection‐Specific Neuronal Populations Using Combinatorial Viral Approaches
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- Abstract
- Table of Contents
- Materials
- Figures
- 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
Table of Contents
- 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
Materials
Basic Protocol 1: Using Combinatorial Viral Strategies to Study Gene‐Circuit Interfaces
Materials
Support Protocol 1: Stereotaxic Surgery for Viral Delivery to Brain
Materials
Support Protocol 2: Adeno‐Associated Virus Production
Materials
Support Protocol 3: Production of CAV2‐Cre
Materials
Support Protocol 4: Brain Sectioning
Materials
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Figures
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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. View Image -
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). View Image
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