Sequencing was performed at the UC Davis DNA Technologies Core. We thank the lab of Lin Tian at UC Davis for training on rAAV packaging and generously gifting us AAV helper and rep/cap plasmids. This work was supported by NIH/NIGMS R35GM119831.

The authors declare no competing financial interests.

This protocol&#160;describes an rAAV-based method for the deployment of enhancer-driven transgenes in the postnatal mouse brain. In this generalized protocol, a candidate enhancer, a minimal promoter, a reporter gene, and an optional barcode sequence are cloned into an AAV plasmid backbone. These experiments can be done with a single candidate enhancer sequence or with many sequences in parallel. The plasmid is packaged into an rAAV and delivered to the postnatal mouse brain. After a period of time to allow for virus tra...

Enhancers are genomic cis-regulatory elements that serve as transcription factor binding sites and can drive the expression of a target gene in a spatiotemporally specific manner1,2. They are differentially active in different cell types, tissues, and stages of development and can be substrates of disease risk-related genomic variation3,4. Thus, the need to understand the dynamics of enhancer function is critical to progress in both translational and basic science applications within genomics. In silico predictions of enhancer activity can serve as excellent resources for generating hypotheses as to enhancer capability5,6. Such predicted enhancer activity can require additional validation and interrogation for a full understanding of the functional activity. Enhancer reporter assays have proved valuable for this purpose across a variety of systems, from cells to animals7,8,9. Towards extending these studies in a flexible and cost-effective transient in vivo context, this protocol describes the use of in vivo AAV-based methods to test putative enhancer sequences for their ability to drive the expression of an ectopic reporter gene in the postnatal mouse brain. This family of methods has utility for interrogating single candidate sequences or parallel library screening and is relevant for basic and translational research.
This method combines in a single plasmid a putative enhancer candidate DNA sequence with a reporter gene (here EGFP), under the control of a minimal promoter that alone does not drive significant expression. The plasmid is packaged into recombinant AAV (rAAV) and injected into an animal model. While the application here is to the brain, various rAAV serotypes enable infection across different tissue types so that this approach can be extended to other systems10. After a period of time, the brain can be collected and assayed for the expression of the reporter gene. Strong expression, compared with controls, indicates that the tested candidate sequence was able to &#34;enhance&#34; the expression of the gene (Figure 1). This simple design offers an easy and clear approach to test a sequence for enhancer activity in vivo in the brain.
In addition to testing for enhancer capability of a sequence, this method can be combined with techniques to determine cell-type enhancer activity. In sequence-based approaches to determining differential enhancer activity, sorting cells on cell-type-specific markers prior to DNA and RNA sequencing can allow researchers to determine if different cell types show differential enhancer activity, as was described in Gisselbrecht et al.11. In imaging-based approaches, co-labeling images with cell-type-specific markers allows examination of&#160;whether cells exhibiting enhancer-driven fluorescence also display cell-type markers of interest12,13,14,15,16. Enhancer reporter assays enable direct testing of risk-associated allelic variation in enhancers for effects on enhancer capability. The vast majority of risk loci identified in genome-wide association studies (GWAS) lie in non-coding regions of the genome17. Functional annotation studies of these risk loci indicate that a large portion likely act as enhancers18,19,20. MPRA deployment in vivo can allow testing of these risk-associated variants for enhancer activity in brain12,21. Finally, delivery and collection at different time points can offer insights into the developmental stages during which an enhancer is active.
Enhancer-reporter plasmid designs are diverse and can be customized to suit experimental goals. There are several options for minimal promoters that have been used in enhancer research, such as the human &#946;-globin minimal promoter22 and the mouse Hsp68 minimal promoter23. These promoters are known to drive low levels of expression unless coupled with an enhancer element to activate them. In contrast, constitutive promoter elements drive strong expression of the transgene, useful for positive control or to test for enhancer function against a background of robust expression. Common choices for constitutive promoters include CAG, a hybrid promoter derived from the chicken &#946;-actin promoter and the cytomegalovirus immediate-early enhancer24, or human EF1&#945;25. Since enhancers are known to work bidirectionally26, the orientation and location of the enhancer relative to the minimal promoter are flexible (Figure 2A). Traditional enhancer-reporter assays place the enhancer upstream of the promoter and, in library deliveries, include a barcode sequence downstream of the reporter gene to associate sequencing reads with the tested enhancer27. However, enhancers can also be placed in the open reading frame of the reporter gene and serve as their own barcode sequence, as is done in STARR-seq28. The protocol described here utilizes the STARR-seq assay design, placing the candidate enhancer sequence into the 3&#39; UTR of the reporter gene. While the STARR-seq orientation offers the benefit of more streamlined cloning, it is less well understood than the conventional approach and may induce variable RNA stability between constructs. The described methods can be easily adapted to either the STARR-seq or conventional orientation with minor alterations to the cloning process that have been described elsewhere27,29.
Different methods of AAV delivery can be employed to further customize this technique to fit experimental goals (Figure 2B). Direct intracranial injections, described further in this protocol, deliver a high concentration of virus directly to the brain30. This gives a high transduction efficiency centered at the site of injection, making this an excellent technique for experiments looking to maximize the density of transduced cells over an area of tissue. Stereotactic injection can help standardize the site of injection across animals for reproducible localized transduction. Intracranial injections are most straightforward in early postnatal animals. As an alternative technique, systemic injections can deliver transgenes using AAVs with serotypes capable of crossing the blood-brain barrier31. Tail-vein injections allow the virus to circulate throughout the body, enabling generalized delivery across many tissues10. Retro-orbital injections are another systemic injection technique that delivers the virus behind the eye into the retro-orbital sinus32. This offers a more direct route for the AAV from the venous system to the brain, resulting in a higher concentration of transduced cells in the brain than injections into more peripheral blood vessels33.
Another flexible aspect of this technique is the method of readout. Broadly, options can be described as reporter-based or sequencing-based (Figure 2C). Incorporating a fluorescent reporter such as GFP into the open reading frame of the construct results in the expression of the fluorescent protein in any transduced cells where the candidate enhancer drove expression. Labeling and imaging techniques such as immunohistochemistry enable signal amplification. Sequencing-based readout techniques involve identifying sequences from the delivered construct in RNA collected from the tissue. By quantifying the amount of viral DNA that was initially delivered, the comparison of expressed RNA versus delivered DNA can be used to determine the degree to which a tested enhancer sequence was capable of driving increased expression of the transgene, for example, in the context of a massively parallel reporter assay (MPRA). MPRAs offer a powerful expansion of these techniques to test up to thousands of candidate enhancers for activity simultaneously and have been described extensively in genomics research12,27,34,35,36. Higher throughput screening is achieved by executing cloning, packaging, delivery, and sequencing steps for candidate enhancers in batch rather than individually.
Candidate enhancer selection provides another opportunity for flexibility (Figure 2D). For example, this assay can be used to identify enhancers of a specific gene, to ascertain the function of non-coding DNA regions of interest, or to determine specific cell types or developmental stages during which an enhancer is active - all of which serve goals both in basic science and in disease research. Generally, candidate enhancer selection is driven by in silico predictions of enhancer activity. Commonly, in silico predictions include ChIP-seq for histone modifications that indicate likely enhancers, such as H3K27ac37 and chromatin accessibility mapping38. Finally, a growing area of research is the function-based screening of synthetically designed enhancer elements, enabling studies of how enhancer sequence directs function39 and the design of enhancers with specific properties40.

<table><tbody><tr><td>10x Citrate Buffer</td><td>Sigma-Aldrich</td><td>C9999-1000ML</td><td></td></tr><tr><td>5&#039;-gatcactctcggcatggac-3&#039;</td><td>Integrated DNA Technologies</td><td>N/A: Custom designed</td><td>Forward primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.</td></tr><tr><td>5&#039;-gatggctggcaactagaagg-3&#039;</td><td>Integrated DNA Technologies</td><td>N/A: Custom designed</td><td>Reverse primer for verifying clones after transformation. These primers are specific to the vector used and were designed for the specific vector used in our experiments.</td></tr><tr><td>Agarose</td><td>VWR</td><td>VWRVN605-500G</td><td></td></tr><tr><td>Aspirator tube assemblies</td><td>Sigma-Aldrich</td><td>A5177-5EA</td><td>for mouth-driven delivery of rAAV</td></tr><tr><td>Bacteriological petri dishes</td><td>Thermo Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Carbenicillin</td><td>Sigma-Aldrich</td><td>C1389-5G</td><td></td></tr><tr><td>Chicken IgY anti-GFP</td><td>Thermo Fisher Scientific</td><td>A10262</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM900</td><td>The images were taken on the LSM800 model, but Zeiss launched the LSM900 model in recent years to replace LSM800.</td></tr><tr><td>Conical centrifuge tubes 15 mL</td><td>Thermo Fisher Scientific</td><td>12-565-269</td><td></td></tr><tr><td>Cryomolds</td><td>Thermo Fisher Scientific</td><td>NC9806558</td><td>These molds are suitable for P28 mouse brain. Other sizes may be more suitable for larger or smaller tissues.</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542-10MG</td><td></td></tr><tr><td>Dissecting scissors, 4.5&quot;</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Donkey anti-chicken AlexaFlour-488</td><td>Jackson ImmunoResearch</td><td>703-545-155</td><td></td></tr><tr><td>Dulbecco&#039;s PBS 1x</td><td>Thermo Fisher Scientific</td><td>MT21031CV</td><td></td></tr><tr><td>Eppendorf Microcentrifuge tubes 2.0 mL</td><td>Thermo Fisher Scientific</td><td>22431048</td><td></td></tr><tr><td>Falcon round-bottom tubes 14 mL</td><td>Thermo Fisher Scientific</td><td>352059</td><td></td></tr><tr><td>Fast Green dye</td><td>Grainger</td><td>F0099-1G</td><td></td></tr><tr><td>Fine detail paint brush set</td><td>Artbrush Tower</td><td>B014GWCLFO</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611S</td><td></td></tr><tr><td>Glass capillary tubes</td><td>Drummond Scientific Company</td><td>5-000-2005</td><td></td></tr><tr><td>HiSpeed Plasmid Maxi Kit</td><td>QIAGEN</td><td>12663</td><td>Commercial plasmid maxi prep kit</td></tr><tr><td>HyClone HyPure Molecular Biology Grade Water</td><td>VWR</td><td>SH30538.03</td><td></td></tr><tr><td>IV butterfly infusion set with 12&quot; tubing and 25G needle</td><td>Thermo Fisher Scientific</td><td>26708</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly Clark</td><td>34155</td><td>Lint-free wipe</td></tr><tr><td>LB Agar</td><td>Thermo Fisher Scientific</td><td>BP1425-500</td><td>LB agar pre-mix for selective media</td></tr><tr><td>McPherson Vannas iris scissor</td><td>Integra LifeSciences</td><td>360-215</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma Life Science</td><td>69794-500ML</td><td></td></tr><tr><td>NEB Stable Competent E. coli</td><td>NEB</td><td>C3040I</td><td></td></tr><tr><td>NucleoSpin Gel and PCR Clean-Up</td><td>Takara</td><td>740609.5</td><td>Kit for enzymatic reaction cleanup and gel extraction</td></tr><tr><td>OCT medium</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>Orbital shaker</td><td>Cole Parmer</td><td>60-100</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127-500G</td><td></td></tr><tr><td>PCR strip tubes 0.2 mL</td><td>VWR</td><td>490003-692</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson</td><td>F155005</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 10x</td><td>Thermo Fisher Scientific</td><td>70011044</td><td></td></tr><tr><td>Phusion Hot Start II High Fidelity DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>F549L</td><td></td></tr><tr><td>Powdered milk</td><td>Sunny Select</td><td></td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>P36934</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28106</td><td></td></tr><tr><td>rCutSmart Buffer</td><td>NEB</td><td>B6004S</td><td>Buffer for restriction digest with PacI, AscI, and XmaI</td></tr><tr><td>Restriction enzyme: AscI</td><td>NEB</td><td>R0558L</td><td></td></tr><tr><td>Restriction enzyme: PacI</td><td>NEB</td><td>R0547L</td><td></td></tr><tr><td>Restriction enzyme: XmaI</td><td>NEB</td><td>R0180L</td><td></td></tr><tr><td>SOC outgrowth medium</td><td>NEB</td><td>B0920S</td><td>Recovery medium after transformation</td></tr><tr><td>Sucrose (RNase/DNase free)</td><td>Millipore Sigma</td><td>033522.5KG</td><td></td></tr><tr><td>TAE buffer</td><td>Apex</td><td>20-194</td><td></td></tr><tr><td>Transfer tubing</td><td>Gilson</td><td>F1179941</td><td>For peristaltic pump</td></tr><tr><td>Triton X100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Wizard Plus SV Minipreps DNA Purification System</td><td>Thermo Fisher Scientific</td><td>A1460</td><td>Plasmid mini prep kit</td></tr></tbody></table>

aav deployment of enhancer-based expression constructs in vivo in mouse brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific genes. Multiple means of assessing non-coding DNA and various chromatin states have proven useful in predicting the presence of enhancer sequences in the genome, but validating the activity of these sequences and finding the organs and developmental stages they are active in is a labor-intensive process. Recent advances in adeno-associated virus (AAV) vectors have enabled the widespread delivery of transgenes to mouse tissues, enabling in vivo enhancer testing without necessitating a transgenic animal. This protocol shows how a reporter construct that expresses EGFP under the control of a minimal promoter, which does not drive significant expression on its own, can be used to study the activity patterns of candidate enhancer sequences in the mouse brain. An AAV-packaged reporter construct is delivered to the mouse brain and incubated for 1-4 weeks, after which the animal is sacrificed, and brain sections are observed under a microscope. EGFP appears in cells in which the tested enhancer is sufficient to initiate gene expression, pinpointing the location and developmental stage in which the enhancer is active in the brain. Standard cloning methods, low-cost AAV packaging, and expanding AAV serotypes and methods for in vivo delivery and standard imaging readout make this an accessible approach for the study of how gene expression is regulated in the brain.

Using these methods, a 915 bp sequence in the psychiatric risk-associated third intron of the gene CACNA1C19,49,50&#160;was tested for enhancer activity in the postnatal mouse brain. This sequence was discovered in an MPRA of 345 candidate enhancer sequences centered on psychiatric and neurological risk SNPs12 and characterization experiments are described here as a general example. C57BL/6 mice ...

Watch this Scientific Journal Video about AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain at JoVE.com

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Enhancers are binding platforms for a diverse array of transcription factors that drive specific expression patterns of tissue- and cell-type-specific ...

aav-deployment-enhancer-based-expression-constructs-vivo-mouse

Research

JoVE Journal

Neuroscience

2.6K Views.  University of California, Davis. This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

Video: AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

AAV Systems and Mouse Models to Study Ectopic Neurod1 Expression Post-Stroke

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.

This protocol describes the viral-mediated ectopic expression of Neurod1 following cortical ischemic stroke. Neurod1 is delivered (1) using the Cre-Flex AAV system in wild-type mice during the subacute phase post-stroke (7 days) and (2) using a single AAV vector in conditional reporter mice during the chronic phase post-stroke (21 days).

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic ...

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain

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Chapters in this video

Production and Titering of Recombinant Adeno-associated Viral Vectors

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke