Name: mosaic analysis of gene function in postnatal mouse brain development by using virus-based cre recombination
Uploaded: 2011-08-01T12:00:00
Description: Watch this Scientific Journal Video about Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination at JoVE.com

This work is supported by an RO1 grant from NIH (NINDS).

1. Preparing viruses for injection
<ol>
 <li>rAAVs were purchased from the recommended commercial vendor, but they can also be produced in one's own lab (see discussion below). The virus solution is typically produced at a titer of &tilde;1x1012 genome copies per milliliter (GC/ml) and may be used at full titer to manipulate a large number of cells. Alternatively, they may be diluted to produce the desired level of sparse labeling. The appropriate dilution must be determined by the user, but a 1:10 dilution ...

<ol>
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The neonatal viral injection method presented here provides a simple and rapid way to generate in vivo mosaics for the study of postnatal brain development. The method takes advantage of floxed alleles that are currently available as well as those that are being made through the High Throughput Gene Targeting project6. Compared to the use of transgenic expression of Cre, this method provides a rapid way to test gene function in various cell types, as mice carrying the floxed alleles can be used dire...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>rAAV8-Cre, -GFP, -DsRed,</td>
			<td>Vector Biolabs</td>
			<td>#7060, #7061, custom order</td>
			<td>http://www.vectorbiolabs.com</td>
		</tr>
		<tr>
			<td>Harvard Pump 11 Plus</td>
			<td>Harvard Apparatus</td>
			<td>#702208</td>
			<td>(No foot pedal port)</td>
		</tr>
		<tr>
			<td>Retinal Pigment Epithelium Injection Kit</td>
			<td>World Precision Instruments</td>
			<td>RPE-KIT</td>
			<td>Contains connective tubing, injection needles (36G), and needle holder</td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 100&mu;l</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL-100</td>
			<td>Includes reusable loading needle</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Invitrogen</td>
			<td>14040-117</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>VWR</td>
			<td>97042-610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROSA26R mouse</td>
			<td>Jackson Laboratory</td>
			<td>003309</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination at JoVE.com

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

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 ...

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