Name: efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor
Uploaded: 2021-11-30T14:45:00
Description: Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

本研究获浸会大学种子基金及2级启动资助（RG-SGT2/18-19/SCI/009）、研究资助局合作研究基金（CRF-C2103-20GF）资助予C.H.H. Hor。

所有与动物有关的程序均按照动物处理指引及香港卫生署批准的规程进行。根据《动物（实验控制）条例》（第340章）取得动物实验牌照，已向香港政府卫生署索取。动物工作是按照香港浸会大学研究处及实验室安全委员会批准的动物安全道德规范进行的。有关此协议中使用的所有材料的详细信息，请参阅材料 表 。
1. 体验前准备
<ol><li>培养基和缓...

<ol>
	<li>Chang, C. H., 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=Atoh1+controls+primary+cilia+formation+to+allow+for+SHH-triggered+granule+neuron+progenitor+proliferation.">Atoh1 controls primary cilia formation to allow for SHH-triggered granule neuron progenitor proliferation.</a> Developmental Cell. 48 (2), 184-199 (2019).</li><li>Dahmane, N., Ruizi Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic Hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126 (14), 3089-3100 (1999).</li><li>Lewis, P. M., Gritti-Linde, A., Smeyne, R., Kottmann, A., Mcmahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+signaling+is+required+for+expansion+of+granule+neuron+precursors+and+patterning+of+the+mouse+cerebellum.">Sonic Hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum.</a> Developmental Biology. 270 (2), 393-410 (2004).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22 (1), 103-114 (1999).</li><li>Bangs, F., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+and+mammalian+Hedgehog+signaling.">Primary cilia and mammalian Hedgehog signaling.</a> Cold Spring Harbor Perspectives in Biology. 9 (5), 028175 (2017).</li><li>Hor, C. H. H., Lo, J. C. W., Cham, A. L. S., Leung, W. Y., Goh, E. L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+functions+of+Rab23+on+primary+cilium-+and+Hedgehog+signaling-mediated+cerebellar+granule+cell+proliferation.">Multifaceted functions of Rab23 on primary cilium- and Hedgehog signaling-mediated cerebellar granule cell proliferation.</a> Journal of Neuroscience. 41 (32), 6850-6863 (2021).</li><li>Han, Y. 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=Dual+and+opposing+roles+of+primary+cilia+in+medulloblastoma+development.">Dual and opposing roles of primary cilia in medulloblastoma development.</a> Nature Medicine. 15 (9), 1062-1065 (2009).</li><li>Spassky, N., 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=Primary+cilia+are+required+for+cerebellar+development+and+Shh-dependent+expansion+of+progenitor+pool.">Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool.</a> Developmental Biology. 317 (1), 246-259 (2008).</li><li>Wang, T., Larcher, L., Ma, L., Veedu, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+screening+of+commonly+used+commercial+transfection+reagents+towards+efficient+transfection+of+single-stranded+oligonucleotides.">Systematic screening of commonly used commercial transfection reagents towards efficient transfection of single-stranded oligonucleotides.</a> Molecules. 23 (10), 2564 (2018).</li><li>Chicaybam, 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=An+efficient+electroporation+protocol+for+the+genetic+modification+of+mammalian+cells.">An efficient electroporation protocol for the genetic modification of mammalian cells.</a> Frontiers in Bioengineering and Biotechnology. 4, 99 (2017).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Chiara Manzini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> Journal of Visualized Experiments: JoVE. (23), e990 (2009).</li><li>Mizoguchi, T., 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=Impaired+cerebellar+development+in+mice+overexpressing+VGF.">Impaired cerebellar development in mice overexpressing VGF.</a> Neurochemical Research. 44 (2), 374-387 (2019).</li><li>Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+nerve+cells.">Confocal imaging of nerve cells.</a> Current Laboratory Methods in Neuroscience Research. , 235-247 (2014).</li><li>Corbit, K. 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=Vertebrate+Smoothened+functions+at+the+primary+cilium.">Vertebrate Smoothened functions at the primary cilium.</a> Nature. 437 (7061), 1018-1021 (2005).</li></ol>

通过电穿孔法转染原代GCP培养物中的转染基因通常与细胞活力低和转染效率差有关9，10。本文介绍了一种具有成本效益且可重复的电穿孔方案，该协议已被证明具有高效率和可行性。此外，我们还展示了一种研究原代GCP细胞中原代纤毛依赖性Hh信号通路的简单方法。
其他常见的电穿孔方法通常需要昂贵的细胞类型特异性电穿孔试剂?...

小脑GCP由于其在体内对Hh信号通路的高丰度和高敏感性，被广泛用于研究神经元祖细胞类型中Hh信号通路的机制1，2，3，4。在GCP中，原发纤毛充当关键的Hh信号转导中枢5，协调前体细胞的增殖6，7，8。原发性纤毛上Hh信号传导成分的体外可视化通常具有挑战性，因为它们的内源性基础水平较低。因此，蛋白质表达水平的转基因修饰和目标基因的荧光团标记是在分子分辨率下研究该途径的有用方法。然而，使用基于脂质体的转染方法对 GCP 原代培养物进行遗传操作通常会导致转染效率低下，从而阻碍进一步的分子研究9。电穿孔可提高效率，但通常需要过多的供应商专用和电池类型受限的电穿孔试剂10。
本文介绍了一种高效且具有成本效益的电穿孔方法，用于操纵基仕伯原代培养物中的Hh信号通路组分。使用这种改良的电穿孔方案，绿色荧光蛋白（GFP）标记的平滑转基因（pEGFP-Smo）有效地递送到GCP，并实现了高细胞存活率和转染率（80-90%）。此外，如免疫细胞化学染色所证明的那样，转染的GCP对平滑激动剂诱导的Hh信号通路激活表现出高度敏感性，方法是将EGFP-Smo转运到原代纤毛。该协议应直接适用于涉及难以转染的细胞类型的 体外 遗传修饰的实验，例如人类和啮齿动物原代细胞培养物，以及人类诱导多能干细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GCP Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies LTD</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 70 &#181;m</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I from bovine pancreas</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Earle&#8217;s Balanced Salt Solution</td>
			<td>Gibco, Life Technologies</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, qualified</td>
			<td>Thermo Scientific</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutamMAXTM-I ,100x</td>
			<td>Gibco, Life Technologies</td>
			<td>35050061</td>
			<td>L-glutamine substitute</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco, Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain,suspension</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine Hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Cayman Chemical</td>
			<td>11914-1</td>
			<td>Smoothened agonist</td>
		</tr>
		<tr>
			<td>IF staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP-goat ab</td>
			<td>Rockland</td>
			<td>600-101-215</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Arl13b mouse monoclonal ab</td>
			<td>NeuroMab</td>
			<td>75-287</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Anti-Pax6 rabbit polyclonal ab</td>
			<td>Covance</td>
			<td>PRB-278P</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Secondary antibody mix</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-goat IgG</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 donkey anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-31573</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62247</td>
			<td>Dilution Factor: 1 : 1000</td>
		</tr>
		<tr>
			<td>Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CU 500 cuvette chamber</td>
			<td>Nepagene</td>
			<td>CU500</td>
			<td></td>
		</tr>
		<tr>
			<td>EPA Electroporation cuvette (2 mm gap)</td>
			<td>Nepagene</td>
			<td>EC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies LTD</td>
			<td>31985070</td>
			<td>reduced-serum medium for transfection</td>
		</tr>
		<tr>
			<td>pEGFP-mSmo</td>
			<td>Addgene</td>
			<td>25395</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation</td>
			<td>Nepagene</td>
			<td>NEPA21</td>
			<td>electroporator</td>
		</tr>
	</tbody>
</table>

efficient and cost effective electroporation method to study primary cilium-dependent signaling pathways in the granule cell precursor

原代纤毛是一种关键的信号细胞器，几乎存在于从细胞表面转导刺猬（Hh）信号刺激的每个细胞上。在颗粒细胞前体（GCP）中，原代纤毛充当关键信号中心，通过调节Hh信号通路来协调前体细胞增殖。通过对通路成分的 体外 遗传操作来促进原发性纤毛依赖性Hh信号传导机制的研究，以可视化它们对原发性纤毛的动态定位。然而，使用目前已知的电穿孔方法在GCP的初级培养物中转染转基因通常成本高昂，并且通常导致细胞活力低和不良转染效率。本文介绍了一种高效、经济且简单的电穿孔方案，该方案显示出~80-90%的高转染效率和最佳的细胞活力。这是一种简单、可重复且高效的遗传修饰方法，适用于研究原代 GCP 培养物中原发性纤毛依赖性刺猬信号通路。

使用Opti-MEM（见 材料表）作为通用试剂，这种提出的电穿孔方法可以实现〜80-90%的持续高电穿孔效率（图1）。在电穿孔后DIV 2处，通过定量所有配对的盒状蛋白-6（Pax6）表达GCP细胞中绿色荧光阳性细胞的百分比来确定Smo-EGFP载体的电穿孔效率。DMSO和SAG处理基团的电穿孔效率似乎相当（图1 和 表2）。
此外，...

Watch this Scientific Journal Video about Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor at JoVE.com

Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor

在这里，我们提出了一种可重复的 体外 电穿孔方案，用于原代小脑颗粒细胞前体（GCP）的遗传操作，具有成本效益，高效且可行。此外，该协议还展示了一种简单的方法，用于原代GCP细胞中原代纤毛依赖性刺猬信号通路的分子研究。

高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In ...

该协议展示了一种直接的体外遗传修饰方法，用于原代颗粒细胞前体培养物中原代纤毛依赖性信号通路的分子研究。由于当前转染方法的成本、低生存率和低效率，我们引入了一种简单、经济高效且高效的电穿孔技术，用于研究原代 GCP 培养物中纤毛依赖性信号通路。首先，将0.5毫升培养基加入含有涂层盖玻片的24孔培养板的每个孔中，并在二氧化碳培养箱中将其保持在37摄氏度的温度下。将所需数量的细胞移入无菌的1.5毫升微量离心管中，并在室温下以200x G旋转5分钟。弃去上清液并将细胞沉淀重悬于200微升Opti-MEM中。重复此过程两次，以确保管中不存在残留的培养基。设置列出的电穿孔参数。轻轻地移取电穿孔反应以充分混合，并使用长P200移液器尖端将精确体积的100微升混合物转移到两毫米间隙比色皿中。一旦比色皿进入比色皿室，按下电吸管的omega按钮，并通过坚持100微升的精确体积来注意阻抗值。阻抗值的范围应约为 30 和 35。按下开始按钮以启动脉冲。记录读数框上显示的测量电流值（以焦耳为单位）。从腔室中取出比色皿。立即将100微升预热的培养基加入比色皿中，并通过上下移液两到三次来重悬。立即将细胞悬浮液转移到先前制备的24孔板中。在二氧化碳培养箱中以37摄氏度孵育细胞。第二天在荧光显微镜下观察细胞。在本研究中，DMSO和SAG处理组的电穿孔效率似乎相当。原代纤毛标志物 Arl13b 的免疫染色表明，在载体组和 SAG 处理组的培养物中，GCP 在 div-2 处的纤毛化率。纤毛率说明了在电穿孔后div-2时表达在细胞表面上具有初级纤毛的Pax6表达GCP的百分比。该图显示，在SAG治疗后24小时，Pax6表达GCP细胞原发性纤毛轴突上的平滑EGFP定位显着增加，表明原发性纤毛依赖性刺猬信号通路的深度激活。为了实现更高的电穿孔效率，应确保所用质粒的高纯度，并且质粒电穿孔混合物中不存在残留培养基。该协议应直接适用于难以转染的原代培养物和细胞类型（包括神经元和人类诱导的多能干细胞）的体外遗传修饰实验。

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Alternative Signaling Routes in Gene Expression

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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 ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

FIM成像和FIMtrack：两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...