Name: stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains
Uploaded: 2018-07-10T12:30:00
Description: Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

This work was supported by the Ministry of Science and Technology grants MOST104-2311-B-010-010-MY3, MOST106-2321-B-010-012, the National Health Research Institutes grant NHRI-EX106-10429NI, and&#160;the featured Areas Research Center Program grant from the Ministry of Education through Brain Research Center, National Yang-Ming University in Taiwan,&#160;and Postdoctoral Fellowship grants MOST106-2811-B-010-031 (S.-Y.C.), MOST105-2811-B-010-036 and MOST106-2811-B-010-030 (H.-Y.K.).

The animal protocols described here have been approved by the Animal Care and Use Committees of National Yang-Ming University.
1. Preparation of The Holder for Neonatal Pups in The Stereotaxic Apparatus
<ol>
	<li>Make the head tray: cut the bottom of a 1.5 mL centrifuge tube (15 mm long) into the shape that fits the head of neonatal pups by removing 1/5 of the wall of the tube.</li>
	<li>Take a pipette tip box with the right size that fits with the pedestal of stereotaxic apparatus, and remo...

<ol>
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In the present study, we demonstrate a simple and reliable stereotaxic method for injecting AAV viruses into the striatum of neonatal mouse brains. We microinjected AAV-eGFP-Cre viruses into the striatum of Ai14 reporter mice at P2 and then analyzed the reporter gene expression at P14. We found AAV transduced GFP-positive cells throughout the striatum at rostrocaudal levels. Moreover, nearly all GFP-positive cells co-expressed the tdTomato reporter gene in striatal cells, suggesting a successful Cre-loxP DNA recombinatio...

Modern studies of the structure and function of the brain usually require genetic manipulation of specific genes in neuronal cells. To probe the functions of different genes, transgenic mice carrying mutant alleles, including knockout and knock-in alleles have been routinely generated. Stereotaxic brain surgery for adult rodents is a standard method to locally deliver drugs, viruses, tracers and other reagents to specific regions of rodent brains1,2. Applying the stereotaxic brain surgery to transgenic mice permits one to genetically manipulate the gene function and neuronal activity in specific neuronal populations of the mouse brain. The cell type-specific manipulation provides a powerful approach to decipher neuronal functions in complex neural circuits of the brain3,4,5.
Neural development of the nervous system begins at early embryonic stages, and the developmental processes continue after birth until the juvenile period. Postnatal maturation of the nervous system includes the precise synaptic wiring of neural circuits, which is essential for physiological and cognitive functions of the brain6. Therefore, studying developmental events that occur in neonatal time windows is important not only for understanding normal neural development, but it may also provide insights into the pathogenesis of neurodevelopmental and neuropsychiatric disorders7,8. Although the methods of stereotaxic brain surgery for adult rodents are readily available2,9, few protocols are available on the internet for stereotaxic brain surgery in neonatal mice10,11. In fact, stereotaxic microinjections of reagents into the brains of neonatal mouse pups are difficult, because the head of neonatal pup is too fragile to be fixed in the standard stereotaxic apparatus. Nonetheless, the application of stereotaxic brain surgery to transgenic mice is feasible for neonatal mice12. Here, we describe a simple method with a homemade setup to perform stereotaxic brain surgery in newborn mouse pups. We demonstrate that this technique allows one to conditionally delete floxed genes by microinjecting AAV-expressing Cre DNA recombinase into the striatum of reporter gene mice and conditionally floxed transgenic mice. This technique is also applicable to deliver reagents into the neonatal striatum of wild-type mice.

<table border="0" cellpadding="0" cellspacing="0" width="1134">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">30G PrecisionGlide Needle</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 305106</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Chloroform</td>
			<td style="width:273px;">JT Baker</td>
			<td style="width:199px;">9180-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Hamilton MICROLITER Syringe</td>
			<td style="width:273px;">Hamilton&#160;</td>
			<td style="width:199px;">80300</td>
			<td style="width:395px;">30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE20</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE10</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Micro Flow Rate Syringe Pump</td>
			<td style="width:273px;">Longer Precision Pump Co.</td>
			<td style="width:199px;">TJ-2A (Controller) and L0107-2A (Drive Unit)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">25G syringe</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 302105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Fast green</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:199px;">F-7252</td>
			<td style="width:395px;">0.1%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Standard Stereotaxic Instruments</td>
			<td style="width:273px;">RWD Life Science</td>
			<td style="width:199px;">68037</td>
			<td style="width:395px;">Without using 68030 Mouse/Neonatal Rat Adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-FOXP2 antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab16046</td>
			<td style="width:395px;">Rabbit polyclonal to FOXP2, 1:4K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-RFP antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab65856</td>
			<td style="width:395px;">Mouse monoclonal to RFP, 1:1K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">BX63 microscope</td>
			<td style="width:273px;">Olympus</td>
			<td style="width:199px;">BX63</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">LSM 880 confocal microscope</td>
			<td style="width:273px;">Zeiss</td>
			<td style="width:199px;">LSM 880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Goat anti-rabbit conjugated Alexa fluor594</td>
			<td style="width:273px;">Jackson lmmunoReserch Laboratories Inc.</td>
			<td style="width:199px;">111-585-003</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td>
			<td style="width:273px;">Penn Vector Core</td>
			<td style="width:199px;">AV-9-PV1848</td>
			<td style="width:395px;">Lot # CS0987, 5.506x1013 (GC/mL)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.chicken actin-eGFP</td>
			<td style="width:273px;">AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td>
			<td style="width:199px;">N/A</td>
			<td style="width:395px;">1x1014 (GC/ml)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:269px;">B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">007914</td>
			<td>Ai14</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">026259</td>
			<td style="width:395px;">Foxp2fl/fl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Dulbecco&#8217;s phosphate buffered saline</td>
			<td style="width:273px;">Corning&#160;cellgro</td>
			<td style="width:199px;">21-030-CVR</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

For the first set of experiment, we microinjected 200 nL of AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 viruses (AAV-eGFP-Cre, 1/10 dilution in Dulbecco&#39;s phosphate buffered saline) that express the Cre DNA recombinase fused with GFP into P2 striatum of Ai14 mice. The Ai14 mice express tdTomato reporter gene upon Cre-mediated deletion of loxP-flanked (floxed) STOP cassette (Figure 2F). The brains were harvested at P14 for immunostaining of GFP and tdTomato. Many...

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells 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, ...

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