We thank Bakhos A. Tannous, Ph.D., Director of Vector Development and Production in the Neuroscience Center of Massachusetts General Hospital, Charlestown, Massachusetts, for providing us with the rAAV-eGfp vector, and John Whang for technical assistance. This work was supported by grant R01 NS050408 (to J.S.) from the National Institute of Neurological Disorders and Stroke.

The surgical procedure described has been approved by the Institutional Animal Care and Use Committee (IACUC) of Columbia University.
1. Preparation of Equipment and Virus Particle Suspension
<ol>
	<li>Clean and disinfect the equipment, sterilize the surgical instruments and the V notch spikes that will be used to fix vertebra L1.</li>
	<li>Pull and bevel glass pipettes. We use pipettes that have a tip diameter of 40 &mu;m and are beveled at an angle of 20&deg;. Sterilize the glass pipettes....

<ol>
	<li>Lewandoski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+control+of+gene+expression+in+the+mouse.">Conditional control of gene expression in the mouse.</a> Nature Reviews Genetics. 2, 743-755 (2001).</li><li>Couto, L. B., High, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector-mediated+RNA+interference.">Viral vector-mediated RNA interference.</a> Curr. Opin. Pharmacol. 10, 534-542 (2010).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nature Reviews Neuroscience. 4, 353-364 (2003).</li><li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11, 823-836 (2010).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 21848-21853 (2010).</li><li>Lo, L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72, 938-950 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8, 745-752 (2011).</li><li>Tang, X. Q., Heron, P., Mashburn, C., Smith, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sensory+axon+regeneration+in+adult+spinal+cord.">Targeting sensory axon regeneration in adult spinal cord.</a> J. Neurosci. 27, 6068-6078 (2007).</li><li>Cameron, A. A., Smith, G. M., Randall, D. C., Brown, D. R., Rabchevsky, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+intraspinal+plasticity+after+spinal+cord+injury+alters+the+severity+of+autonomic+dysreflexia.">Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.</a> J. Neurosci. 26, 2923-2932 (2006).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> The Journal of Clinical Investigation. 120, 1253-1264 (2010).</li><li>Lutz, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsymptomatic+restoration+of+SMN+rescues+the+disease+phenotype+in+a+mouse+model+of+severe+spinal+muscular+atrophy.">Postsymptomatic restoration of SMN rescues the disease phenotype in a mouse model of severe spinal muscular atrophy.</a> The Journal of Clinical Investigation. 121, 3029-3041 (2011).</li><li>Chen, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsAAV+type+2-mediated+gene+transfer+of+MORS196A-EGFP+into+spinal+cord+as+a+pain+management+paradigm.">dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 20096-20101 (2007).</li><li>South, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+deletion+of+the+NR1+subunit+of+the+NMDA+receptor+in+adult+spinal+cord+dorsal+horn+reduces+NMDA+currents+and+injury-induced+pain.">A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.</a> J. Neurosci. 23, 5031-5040 (2003).</li><li>Tappe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+scaffolding+protein+Homer1a+protects+against+chronic+inflammatory+pain.">Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.</a> Nat. Med. 12, 677-681 (2006).</li><li>Colle, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+intracerebral+delivery+of+AAV5+vector+encoding+human+ARSA+in+non-human+primate.">Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.</a> Human Molecular Genetics. 19, 147-158 (2010).</li><li>Carbajal, K. S., Weinger, J. G., Whitman, L. M., Schaumburg, C. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Transplantation+of+Mouse+Neural+Stem+Cells+into+the+Spinal+Cords+of+Mice+Infected+with+Neurotropic+Mouse+Hepatitis+Virus.">Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus.</a> J. Vis. Exp. (53), e2834 (2011).</li><li>Snyder, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+adeno-associated+viral+vector+serotypes+for+spinal+cord+and+motor+neuron+gene+delivery.">Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.</a> Hum. Gene Ther. 22, 1129-1135 (2011).</li><li>Towne, C., Pertin, M., Beggah, A. T., Aebischer, P., Decosterd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+serotype+6+(rAAV2/6)-mediated+gene+transfer+to+nociceptive+neurons+through+different+routes+of+delivery.">Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.</a> Mol. Pain. 5, 52 (2009).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian.</a> 8, 148-154 (1994).</li></ol>

Stereotaxic vector injection allows targeting spinal cord neurons for applications such as neuronal network mapping based on transsynaptic virus spreading 6,7 or optogenetic dissection 8, axon guidance during regeneration from injury 9,10, or gene therapy for the prevention or treatment of neurodegeneration 11,12. Viral vectors have been used for gene manipulation in the spinal cord to study somatosensory, motor and autonomic pathways 9,10,13-15. The mouse is the mod...

Advanced technologies of conditional gene manipulation in the mouse enable multifaceted approaches to the exploration of synaptic pathways and functional connections in the central nervous system. Transgenes may be regulated by small-molecule effectors such as doxycycline acting on a tetracycline-controlled transactivator, which can be designed to function as a repressor or an activator of gene transcription, or tamoxifen recognizing a mutated ligand-binding domain of the estrogen receptor 1. Irreversible transgene modification is commonly achieved by deoxyribonucleic acid (DNA) recombinases. Cre (causes recombination) and Flp (flippase recombination enzyme) catalyze the excision, inversion or translocation of DNA fragments that are flanked by loxP (locus of crossing x over, P1) or Frt (flippase recognition target) sites, respectively 1. Applications include gene activation or silencing and inducible ribonucleic acid (RNA) interference 2. Conditional expression of fluorescent or enzymatic reporters such as &beta;-galactosidase or alkaline phosphatase can be used to label neurons and examine their topical organization and connectivity 3. Large-scale mutagenesis projects in North America (<a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a>) and Europe (<a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a>) are producing libraries of mouse embryonic stem cell clones with conditional gene targets and traps that will eventually cover the entire mouse genome. Mice generated from these clones may be crossed with an expanding number of mouse lines that express DNA recombinases under promoters or loci specific to a particular population of neurons for selective gene manipulation (<a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index.php</a>).
However, restricting gene manipulation to distinct populations of neurons or particular regions of interest may not be achieved by genetic targeting alone if a promoter specific to the neuron population of interest is not known or not expressed by all neurons in the region of interest. In the spinal cord, experimental designs may require spatial restriction of the gene manipulation to one or two craniocaudal segments. Stereotaxic injection of a viral vector that expresses Cre or Flp allows limiting gene recombination to regions in the spinal cord of mice in which DNA fragments are flanked by loxP or Frt sites, so-called floxed or flrted alleles. Unlike constitutive DNA rearrangement, which would result from crossbreeding the animals with recombinase expressing mice, this strategy also provides temporal control over gene activation or silencing. Viral vectors encoding floxed or flirted transgenes offer a reverse option of gene manipulation in mice expressing the corresponding recombinase downstream of a neuron-specific promoter. Several recombinant vectors with affinity to neurons are available 4. High-capacity (gutless) adenovirus, adeno-associated virus, herpes simplex virus and lentivirus are commonly used neurotropic vectors. Selecting the appropriate virus for a research question is a crucial part of the experimental design. Size of the transgene, delivery route, specificity of the infection to neurons as opposed to glial cells, infection efficacy, inflammatory and toxic side effects need to be considered 4.
Here we describe the stereotaxic injection of a viral vector into the dorsal horn of the spinal cord, a technique that we employ for conditional gene regulation in our research on the neurobiology of pain. The dorsal horn receives afferent input from primary somatosensory neurons including nociceptive neurons. Local interneurons process the information before projection neurons convey it from the dorsal horn to the brain 5. We demonstrate the infection of dorsal horn neurons at spinal segmental level L4 with a neurotropic recombinant adeno-associated virus (rAAV) that expresses enhanced green fluorescent protein (eGfp) under a constitutively active cytomegalovirus promoter.

<table border="1">
	<tbody>
 	<tr>
 	<td>Material Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td></td>
 	 </tr>
 <tr>
 		<td>Spinal base plate</td>
 <td>David Kopf Instruments</td>
 <td>912</td>
 <td></td>
 </tr>
 <tr>
 		<td>Small animal stereotaxic instrument</td>
 <td>David Kopf Instruments</td>
 <td>900</td>
 <td></td>
 </tr>
 <tr>
 		<td>Mouse gas anesthesia head holder</td>
 <td>David Kopf Instruments</td>
 <td>923-B</td>
 <td></td>
 </tr>
 <tr>
 	<td>Adjustable base mounts</td>
 <td>David Kopf Instruments</td>
 <td>982</td>
 <td></td>
 </tr>
 <tr>
 	<td>V notch spikes</td>
 <td>David Kopf Instruments</td>
 <td>987</td>
 <td></td>
 </tr>
 <tr>
 	<td>Small animal temperature control system</td>
 <td>David Kopf Instruments</td>
 <td>TCAT-2LV</td>
 <td></td>
 </tr>
 <tr>
 	<td>Adson forceps</td>
 <td>Fine Science Tools</td>
 <td>11006-12</td>
 <td></td>
 </tr>
 <tr>
 	<td>Laminectomy forceps</td>
 <td>Fine Science Tools</td>
 <td>11223-20</td>
 <td></td>
 </tr>
 <tr>
 	<td>UltraMicroPump (one) with SYS-Micro4 Controller</td>
 <td>World Precision Instruments</td>
 <td>UMP3-1</td>
 <td></td>
 </tr>
 <tr>
 	<td>Microsyringe, 65RN</td>
 <td>Hamilton</td>
 <td>7633-01</td>
 <td></td>
 </tr>
 <tr>
 	<td>RN compression fitting, 1 mm</td>
 <td>Hamilton</td>
 <td>55750-01</td>
 <td></td>
 </tr>
 <tr>
 	<td>Borosilicate glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td></td>
 </tr>
 <tr>
 	<td>Microgrinder</td>
 <td>Narishige</td>
 <td>EG-44</td>
 <td></td>
 </tr>
 </tbody>
</table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Successful transfection yields robust gene expression in neurons of the injected dorsal horn (Figure 1), sparing the dorsal horn of the contralateral side, the ventral horn and the dorsal root ganglia.
<img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figure 1" /> 
Figure 1. Transfection of dorsal horn neurons. (A) Expression of the fluorescent reporter eGfp (green) in the left dorsal horn of the L4 spinal cord, two weeks after the s...

Watch this Scientific Journal Video about Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord at JoVE.com

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

stereotaxic-injection-viral-vector-for-conditional-gene-manipulation

Research

JoVE Journal

Neuroscience

30.0K visualizações.  Centre National de la Recherche Scientifique (CNRS). Vetores virais permitem a manipulação de genes alvo. Nós demonstramos um método para a expressão de genes condicionais ou ablação da medula espinhal de rato, utilizando a injecção estereotáxica de um vector viral no corno dorsal, um local proeminente de contacto sináptico entre os aferentes primários e os neurónios de somatossensorial no sistema nervoso central.