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

这项工作得到了科学和技术部的支持, MOST104-2311-B-010-010-MY3、MOST106-2321-B-010-012、国家卫生研究所赠款 NHRI-EX106-10429NI 和特色领域研究中心方案赠款来自教育部通过台湾国立阳明大学脑研究中心和博士后奖学金授予 MOST106-2811-B-010-031 (Y.C.)、MOST105-2811-B-010-036 和 MOST106-2811-B-010-030 (H 张佑启)。

这里所描述的动物协议已经得到国立阳明大学动物保育委员会的批准。
1. 在立体定向装置中制备新生儿幼崽的支架
<ol><li>制作头托盘: 将1.5 毫升离心管 (15 毫米长) 的底部切割成适合新生儿幼崽头部的形状, 并除去1/5 的管壁。</li>
	<li>取一个合适大小的吸管尖盒, 适合于立体定向设备的底座, 并取下顶盖。将步骤1.1 中的头托盘和组织嵌入盒贴在热熔胶粘剂的刀尖托盘底座上。?...

<ol>
	<li>Athos, J., Storm, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+precision+stereotaxic+surgery+in+mice.">High precision stereotaxic surgery in mice.</a> Current Protocols in Neuroscience. , A.4A.1-A.4A.9 (2001).</li><li>Cetin, A., Komai, S., Eliava, M., Seeburg, P. H., Osten, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nat. Protoc. 1 (6), 3166-3173 (2006).</li><li>Tye, K. M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+investigation+of+neural+circuits+underlying+brain+disease+in+animal+models.">Optogenetic investigation of neural circuits underlying brain disease in animal models.</a> Nat. Rev. Neurosci. 13 (4), 251-266 (2012).</li><li>Roth, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DREADDs+for+neuroscientists.">DREADDs for neuroscientists.</a> Neuron. 89 (4), 683-694 (2016).</li><li>Knopfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+optical+indicators+for+the+analysis+of+neuronal+circuits.">Genetically encoded optical indicators for the analysis of neuronal circuits.</a> Nat. Rev. Neurosci. 13 (10), 687-700 (2012).</li><li>Tau, G. Z., Peterson, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+development+of+brain+circuits.">Normal development of brain circuits.</a> Neuropsychopharmacol. 35 (1), 147-168 (2010).</li><li>Mitchell, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+neurodevelopmental+disease.">The genetics of neurodevelopmental disease.</a> Curr. Opin. Neurobiol. 21 (1), 197-203 (2011).</li><li>Sahin, M., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes,+circuits,+and+precision+therapies+for+autism+and+related+neurodevelopmental+disorders.">Genes, circuits, and precision therapies for autism and related neurodevelopmental disorders.</a> Science. 350 (6263), (2015).</li><li>Schierberl, K. C., Rajadhyaksha, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+microinjection+of+viral+vectors+expressing+Cre+recombinase+to+study+the+role+of+target+genes+in+cocaine+conditioned+place+preference.">Stereotaxic microinjection of viral vectors expressing Cre recombinase to study the role of target genes in cocaine conditioned place preference.</a> J. Vis. Exp. (77), e50600 (2013).</li><li>Mathon, B., 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=Increasing+the+effectiveness+of+intracerebral+injections+in+adult+and+neonatal+mice:+a+neurosurgical+point+of+view.">Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view.</a> Neurosci. Bull. 31 (6), 685-696 (2015).</li><li>Davidson, S., Truong, H., Nakagawa, Y., Giesler, G. 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+microinjection+technique+for+targeting+regions+of+embryonic+and+neonatal+mouse+brain+in+vivo.">A microinjection technique for targeting regions of embryonic and neonatal mouse brain in vivo.</a> Brain Res. 1307, 43-52 (2010).</li><li>Chen, Y. C., 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=Foxp2+controls+synaptic+wiring+of+corticostriatal+circuits+and+vocal+communication+by+opposing+Mef2c.">Foxp2 controls synaptic wiring of corticostriatal circuits and vocal communication by opposing Mef2c.</a> Nat. Neurosci. 19 (11), 1513-1522 (2016).</li><li>Gerfen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neostriatal+mosaic:+multiple+levels+of+compartmental+organization+in+the+basal+ganglia.">The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia.</a> Annu. Rev. Neurosci. 15, 285-320 (1992).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30 (18), 6171-6177 (2010).</li><li>Cheetham, C. E., Grier, B. D., Belluscio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+regional+viral+injection+in+neonatal+mice+enables+structural+and+functional+interrogation+of+defined+neuronal+populations+throughout+targeted+brain+areas.">Bulk regional viral injection in neonatal mice enables structural and functional interrogation of defined neuronal populations throughout targeted brain areas.</a> Front. Neural Circuit. 9, 72 (2015).</li></ol>

在本研究中, 我们展示了一种简单可靠的立体定向方法, 将 AAV 病毒注入新生小鼠脑纹状体。在 P2, 我们将 AAV eGFP 病毒杏仁 Ai14 小鼠纹状体中, 然后分析了 P14 的报告基因表达。我们发现 AAV 转基因 GFP 阳性细胞在整个纹状体 rostrocaudal 水平。此外, 几乎所有的 GFP 阳性细胞都在巴细胞中共同表达了 tdTomato 报告基因, 这表明 loxP DNA 重组是由 AAV 介导的。我们进一步证实了这种方法的有效性, 通过 microinject...

现代对大脑结构和功能的研究通常需要神经元细胞中特定基因的基因调控。为了探讨不同基因的功能, 转基因小鼠携带突变等位基因, 包括敲除和敲入的基因, 已被常规生成。成人啮齿动物立体定向脑外科是一种标准的方法, 当地提供药物, 病毒, 示踪剂和其他试剂到特定区域的啮齿动物大脑1,2。将立体定向脑外科应用于转基因小鼠, 允许基因调控小鼠大脑特定神经元群中的基因功能和神经元活动。细胞类型特定的操作提供了一个强大的方法来破译神经元功能的复杂神经回路的大脑3,4,5。
神经系统的神经发育始于早期胚胎阶段, 发育过程在出生后持续到幼年期。神经系统的产后成熟包括神经回路的精确突触布线, 这对于大脑6的生理和认知功能是必不可少的。因此, 研究新生儿时间窗发生的发育事件, 不仅对了解正常的神经发育有重要意义, 而且还可以为神经发育和精神疾病的发病机制提供深入的认识7 ,8。虽然成人啮齿目动物的立体定向脑外科手术方法29, 但在互联网上有少量的协议可供新生小鼠10,11的立体定向脑外科手术使用。事实上, 在新生小鼠幼崽的大脑中对试剂进行立体定向 microinjections 是困难的, 因为新生儿幼犬的头部太脆弱, 无法固定在标准的立体定向设备中。然而, 在转基因小鼠中应用立体定向脑手术是可行的12。在这里, 我们描述了一个简单的方法, 自制的设置, 进行立体定向脑外科手术的新生鼠幼崽。我们证明, 这种技术允许一个有条件地删除 floxed 基因 microinjecting AAV 表达的 DNA recombinase 到报告基因小鼠纹状体和有条件 floxed 转基因小鼠。该技术也适用于向野生型小鼠的新生纹状体提供试剂。

<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

许多基因是在胚胎大脑中表达的, 其中有些是在出生后大脑中不断表达的。对于这种持续表达的基因, 它们可以调节新生儿大脑的发育过程和/或生理功能。为了研究大脑中特定基因的神经生物学功能, 在脑中灭活基因是必不可少的。在这里, 我们描述了一个简单的立体定向的方法, 以灭活基因表达在转基因小鼠在新生儿时间窗口。AAV eGFP 病毒通过立体定向脑外科术, 杏仁于产后 (P) 2 Ai14 报告基因小鼠纹状体。在 P14 纹状体中检测到 tdTomato 报告基因表达, 提示 AAV 转基因细胞中成功的 loxP 介导的 DNA 重组。我们进一步验证了这一技术, 通过 microinjecting AAV eGFP 病毒的 P2Foxp2佛罗里达州/佛罗里达州小鼠。荧光蛋白和 Foxp2 的双重标记表明, gfp 阳性细胞在 P9 纹状体中缺乏 Foxp2 免疫反应, 提示 AAV eGFP 细胞 Foxp2 蛋白质的丢失。这些结果表明, 在 floxed 转基因小鼠的新生儿脑中, stereotaxically 杏仁 AAV eGFP 病毒对特定神经元群有有效的遗传缺失。总之, 我们的立体定向技术为新生小鼠大脑中的基因操作提供了一个简单的平台。该技术不仅可以用于删除新生儿大脑特定区域的基因, 而且还可用于注射药理药物、神经元示踪剂、基因修饰光遗传学和 chemogenetics 蛋白、神经元活动指标和其他试剂进入新生鼠脑纹状体。

在第一组实验中, 我们杏仁了 200 AAV9 的 hSynapsin. eGFP WPRE SV40 病毒 (AAV eGFP, 1/10 Dulbecco 磷酸盐缓冲盐水稀释), 表达了与 GFP 的 DNA recombinase 与绿色荧光蛋白融合为 P2 小鼠的 Ai14 纹状体。Ai14 小鼠在 tdTomato 的 loxP 侧 (floxed) 停止盒 (图 2F) 的基础上, 表达了对该基因的报道。大脑是在 P14 染色的 GFP 和 tdTomato 中收获的。许多 AAV 转基因 GFP 阳性细胞存在于整个?...

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

新生小鼠脑巴细胞基因调控的立体定向手术

我们描述了一个立体定向手术的协议, 一个自制的头固定装置 microinjecting 试剂进入新生鼠脑纹状体。这种技术允许在新生小鼠大脑特定区域的神经细胞中进行基因操作。

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

Research

JoVE Journal

Neuroscience

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

科研

神经科学

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

特定脑区的兴奋毒性损伤的成年大鼠立体定向手术治疗

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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

立体定向注射病毒载体的基因操作在小鼠脊髓条件

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

在普通马莫塞特中植入微电极阵列的立体外科（Callithrix jacchus）

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...