Name: direct gene knock-out of axolotl spinal cord neural stem cells via electroporation of cas9 protein-grna complexes
Uploaded: 2019-07-09T14:00:00
Description: Watch this Scientific Journal Video about Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes at JoVE.com

我们感谢田中美利教授的持续和长期支持。这项工作得到了国家自然科学基金(国家自然科学基金)资助(317716)、华南师范大学研究启动补助金(S82111和8S0109)和中国博士后科学基金资助(2018M633067)的支持。

所有动物实验必须按照地方和国家动物实验条例进行,并经有关机构审查委员会批准。
1. 准备CAS9-gRNA RNP组合
<ol><li>设计和合成gRNA。 注:请参阅其他出版物,用于设计和合成gRNA,包括一份专门涉及axolotls 15、20、21、22的出版物。</li>
	<li>通过内部制备或商业购买获得CAS9-NLS蛋白质。<...

<ol>
	<li>O'Hara, C. M., Egar, M. W., Chernoff, E. 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=Reorganization+of+the+ependyma+during+axolotl+spinal+cord+regeneration:+Changes+in+intermediate+filament+and+fibronectin+expression.">Reorganization of the ependyma during axolotl spinal cord regeneration: Changes in intermediate filament and fibronectin expression.</a> Developmental Dynamics. 193 (2), 103-115 (1992).</li><li>Mchedlishvili, L., Mazurov, V., Tanaka, 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=Reconstitution+of+the+Central+Nervous+System+During+Salamander+Tail+Regeneration+from+the+Implanted+Neurospheres.">Reconstitution of the Central Nervous System During Salamander Tail Regeneration from the Implanted Neurospheres.</a> Plant, Soil and Environment. 916 (8), 197-202 (2012).</li><li>Nordlander, R. H., Singer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ependyma+in+regeneration+of+the+spinal+cord+in+the+urodele+amphibian+tail.">The role of ependyma in regeneration of the spinal cord in the urodele amphibian tail.</a> Journal of Comparative Neurology. 180 (2), 349-373 (1978).</li><li>Fei, J. F., 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=Tissue-+and+time-directed+electroporation+of+CAS9+protein&#8211;gRNA+complexes+in+vivo+yields+efficient+multigene+knock-out+for+studying+gene+function+in+regeneration.">Tissue- and time-directed electroporation of CAS9 protein&#8211;gRNA complexes in vivo yields efficient multigene knock-out for studying gene function in regeneration.</a> npj Regenerative Medicine. 1 (1), 16002 (2016).</li><li>Fei, J. F., 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=CRISPR-mediated+genomic+deletion+of+Sox2+in+the+axolotl+shows+a+requirement+in+spinal+cord+neural+stem+cell+amplification+during+tail+regeneration.">CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration.</a> Stem Cell Reports. 3 (3), 444-459 (2014).</li><li>Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+targeted+mutagenesis+in+axolotl+using+Cas9+RNA-guided+nuclease.">Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease.</a> Development. 141 (10), 2165-2171 (2014).</li><li>Flowers, G. P., Sanor, L. D., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+of+genome-edited+alleles+reveals+high+fidelity+axolotl+limb+regeneration.">Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration.</a> eLife. 6, (2017).</li><li>Fei, J. -. F., 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+gene+knockin+in+axolotl+and+its+use+to+test+the+role+of+satellite+cells+in+limb+regeneration.">Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.</a> Proceedings of the National Academy of Sciences. 201706855. , (2017).</li><li>Nowoshilow, 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=The+axolotl+genome+and+the+evolution+of+key+tissue+formation+regulators.">The axolotl genome and the evolution of key tissue formation regulators.</a> Nature. 554 (7690), 50-55 (2018).</li><li>Bryant, D. 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+Tissue-Mapped+Axolotl+De+Novo+Transcriptome+Enables+Identification+of+Limb+Regeneration+Factors.">A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors.</a> Cell Reports. 18 (3), 762-776 (2017).</li><li>Smith, J. J., 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+chromosome-scale+assembly+of+the+axolotl+genome.">A chromosome-scale assembly of the axolotl genome.</a> Genome Research. 373548, (2019).</li><li>Smith, J. 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J., 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=et+al+Gene+expression+profile+of+the+regeneration+epithelium+during+axolotl+limb+regeneration.">et al Gene expression profile of the regeneration epithelium during axolotl limb regeneration.</a> Developmental Dynamics. 240 (7), 1826-1840 (2011).</li><li>Stewart, 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=Comparative+RNA-seq+Analysis+in+the+Unsequenced+Axolotl:+The+Oncogene+Burst+Highlights+Early+Gene+Expression+in.">Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in.</a> the Blastema. PLoS Computational Biology. 9 (3), (2013).</li><li>Fei, J. -. F., 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=Application+and+optimization+of+CRISPR&#8211;Cas9-mediated+genome+engineering+in+axolotl+(Ambystoma+mexicanum).">Application and optimization of CRISPR&#8211;Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum).</a> Nature Protocols. 13 (12), 2908-2943 (2018).</li><li>Holder, 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=Continuous+growth+of+the+motor+system+in+the+axolotl.">Continuous growth of the motor system in the axolotl.</a> Journal of Comparative Neurology. 303 (4), 534-550 (1991).</li><li>Tazaki, A., Tanaka, E. M., Fei, J. 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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=et+al+Planar+cell+polarity-mediated+induction+of+neural+stem+cell+expansion+during+axolotl+spinal+cord+regeneration.">et al Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration.</a> eLife. 4, 1-29 (2015).</li><li>Cong, 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Doench, J. 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所述协议允许在轴线脊髓的NSC中进行时间和空间限制基因敲除。当前协议允许在定义的时间和位置对 NSC 进行特定定位,且渗透性较高。它避免了在使用 Cre-LoxP 系统时在其他地区(如大脑)中来自 NSC 中基因敲除的潜在不良效应。它还避免了来自持续淘汰的发育影响,允许研究以再生为重点的基因功能。此外,该协议可以在野生型动物中执行,避免产生Cre-LoxP系统所需的额外转基因轴胶所需的长时间等待。...

大多数脊椎动物的脊髓在受伤后无法再生,导致永久性残疾。几个蜥蜴,如异种,是明显的例外。axolotl 可以完全再生结构相同的脊髓,并完全恢复脊髓功能。轴线脊髓的再生能力大部分是由于脑膜细胞。这些细胞排列在中央通道上,与哺乳动物不同,轴球细胞作为神经干细胞(NSC)在胚胎后发育。脊髓损伤(例如,从尾部截肢)后,这些NSC增殖,以重新生长的脑管和分化,以取代丢失的神经元1,2,3。揭示轴线脊髓NSC如何保持多能和在损伤后激活,可以为人类患者开发新的治疗策略提供有价值的信息。
由于CRISPR-Cas9基因敲除技术的进步,执行敲除破译基因功能变得更容易,并已证明在各种物种中具有广泛的适用性,包括axolotls4,5,6,7,8.最近发布的完整 axolotl 基因组和转录组现在允许任何基因组位点被靶向,并更好地评估偏离目标的效果 9,10,11,12,13,14.使用CRISPR-Cas9系统15,为敲除和敲击的轴线开发了优化的协议。以CAS9蛋白-gRNA核糖核蛋白(RNP)的形式交付CRISPR-Cas9机械,已经证明比使用Cas9和gRNA编码质粒4更有效。这可能是由于RNP比质粒载体尺寸小,它能够立即产生DNA断裂,并保护gRNA免受RNA降解。此外,使用 RNP 可绕过转录和翻译;因此,当质粒元素来自不同的物种时,它避免了启动子强度和最佳柯顿使用等问题。
功能丧失研究是研究感兴趣的基因的潜在功能的一般方法之一。为了研究再生过程中的基因功能,最好在受伤前进行敲除,以避免对发育的影响。此外,淘汰应限于 NSC 和再生区域。在所有NSC(包括大脑中的目标基因,即Cre-LoxP系统中)中,目标基因的敲除可能产生与再生无关的效果,从而混淆结果的解释。幸运的是,在NSC中,轴线脊髓的结构为时间和空间受限的淘汰提供了一个独特的机会。大多数脊髓NSC与中央运河接触,构成与中央运河接触的绝大多数细胞16、17。因此,将CAS9-gRNA复合物注射到中央通道,然后进行电穿孔,允许在4、18、19的特定时间将脊髓NSC输送到所需区域。该协议演示了如何执行,导致目标脊髓NSC的高度穿透性敲除。然后进行后续分析,以研究对再生和NSC行为的影响。

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			<td height="28" style="height:28px;width:492px;">Agarose</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">A9539</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">&#65279;Benzocaine</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">E1501-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:492px;">&#65279;Benzocaine 0.03 % (wt/vol)</td>
			<td style="width:455px;">Mix 500 ml of 10&#215; TBS, 500 ml of 400% (wt/vol) Holtfreter&#8217;s solution and 30 ml of 10% (wt/vol) benzocaine stock solution. Fill up the volume to 10 L with dH2O. &#65279;The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
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		<tr height="84">
			<td height="84" style="height:84px;width:492px;">&#65279;Benzocaine 10 % (wt/vol)</td>
			<td style="width:455px;">Mix 50 g of benzocaine in 500 ml of 100% (vol/vol) ethanol. The solution can be stored at room temperature for up to 12 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
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			<td height="28" style="height:28px;width:492px;">Borosilicate glass capillaries &#65279;1.2 mm O.D., 0.94 mm I.D.</td>
			<td style="width:455px;">Stutter Instrument&#160;</td>
			<td style="width:241px;">BF120-94-8</td>
			<td style="width:167px;"></td>
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		<tr height="31">
			<td height="31" style="height:31px;width:492px;">CaCl2&#183;2H2O</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;102382</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:492px;">CAS9 buffer, 10x</td>
			<td style="width:455px;">Mix 200 mM HEPES and 1.5 M KCl in RNase-free water. Adjust pH to 7.5. Filter sterilize, aliquot and store at &#8722;20 &#176;C for up to 24 months</td>
			<td style="width:241px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:492px;">&#65279;CAS9-NLS protein&#160;&#160;</td>
			<td style="width:455px;">PNA Bio</td>
			<td style="width:241px;">CP03</td>
			<td style="width:167px;"></td>
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			<td height="28" style="height:28px;width:492px;">Cell culture dishes, 10cm</td>
			<td style="width:455px;">Falcon</td>
			<td style="width:241px;">351029</td>
			<td style="width:167px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Dumont #5 - Fine Forceps</td>
			<td style="width:455px;">Fine Scientific Instruments</td>
			<td style="width:241px;">11254-20</td>
			<td style="width:167px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Electroporator</td>
			<td style="width:455px;">Nepa Gene&#160;</td>
			<td style="width:241px;">NEPA21</td>
			<td style="width:167px;"></td>
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		<tr height="56">
			<td height="56" style="height:56px;width:492px;"></td>
			<td style="width:455px;">BEX</td>
			<td style="width:241px;">Pulse Generator CUY21EDIT II</td>
			<td style="width:167px;"></td>
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			<td height="28" style="height:28px;width:492px;">Fast Green FCF</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">F7252-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:492px;">Fast Green FCF Solution, 5x</td>
			<td style="width:455px;">Dissolve 12.5 mg of Fast Green FCF powder in 10 mL of 1&#215; PBS.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Flaming/Brown Micropipette Puller&#160;</td>
			<td style="width:455px;">Stutter Instrument&#160;</td>
			<td style="width:241px;">P-97</td>
			<td style="width:167px;"></td>
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		<tr height="105">
			<td height="105" style="height:105px;width:492px;">&#65279;Holtfreter&#8217;s solution 400% (wt/vol)&#160;</td>
			<td style="width:455px;">&#65279;Dissolve 11.125 g of MgSO4&#183;7H2O, 5.36 g of CaCl2&#183;2H2O, 158.4 g of NaCl and 2.875 g of KCl in 10 L of dH2O. The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
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			<td height="28" style="height:28px;width:492px;">KCl</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;104936</td>
			<td style="width:167px;"></td>
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		<tr height="31">
			<td height="31" style="height:31px;width:492px;">MgSO4&#183;7H2O</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;105886</td>
			<td style="width:167px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Microloader pipette tips</td>
			<td style="width:455px;">Eppendorf</td>
			<td style="width:241px;">5242956003</td>
			<td style="width:167px;"></td>
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		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Micromanipulator&#160;</td>
			<td style="width:455px;">Narishige</td>
			<td style="width:241px;">MN-153&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">NaCl</td>
			<td style="width:455px;">Merck</td>
			<td style="width:241px;">&#65279;106404</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Pneumatic PicoPump</td>
			<td style="width:455px;">World Precision Instruments&#160;</td>
			<td style="width:241px;">SYS-PV830</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Ring Forceps</td>
			<td style="width:455px;">Fine Scientific Instruments</td>
			<td style="width:241px;">11103-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Stereomicroscope</td>
			<td style="width:455px;">Olympus</td>
			<td style="width:241px;">SZX10&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:492px;">Tris base</td>
			<td style="width:455px;">Sigma-Aldrich</td>
			<td style="width:241px;">&#65279;T6066</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="112">
			<td height="112" style="height:112px;width:492px;">Tris-buffered saline, 10x</td>
			<td style="width:455px;">&#65279;Dissolve 24.2 g of Tris base and 90 g of NaCl in 990 ml of dH2O. Adjust pH to 8.0 by adding 10 ml of 37% (vol/vol) HCl. The solution can be stored at room temperature for up to 6 months.</td>
			<td style="width:241px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:492px;">Tweezers w/Variable Gap 2 Round Platinum Plate Electrode, 10mm diameter</td>
			<td style="width:455px;">Nepa Gene&#160;</td>
			<td style="width:241px;">&#65279;CUY650P10</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

direct gene knock-out of axolotl spinal cord neural stem cells via electroporation of cas9 protein-grna complexes

轴线具有完全再生脊髓的独特能力。这主要是由于脑细胞作为神经干细胞(NSCs)在一生中残留,这些细胞增殖以改造脑管,并在脊髓损伤后分化成丢失的神经元。解读这些 NSC 如何在脊髓损伤后保持多能性并在脊髓损伤时增殖,以改革确切的损伤前结构,从而对哺乳动物脊髓如何再生以及潜在的治疗方案提供有价值的见解。在限定时间内在NSC的特定子集中执行基因敲除,将使得研究这些再生过程背后的分子机制,而不会被发育扰动效应所迷惑。此处描述的是一种使用 CRISPR-Cas9 系统在 axolotl 脊髓 NSC 中执行基因敲除的方法。通过将 CAS9-gRNA 复合物注入脊髓中央管,然后进行电穿孔,目标基因在脊髓特定区域的 NSC 中按所需时间点被敲掉,从而在脊髓 NSC 期间进行分子研究再生。

CAS9-gRNA复合物对Sox2的注射和电穿孔进入轴球脊髓中央管,导致大多数脊髓NSC中SOX2免疫反应性严重丧失,以gRNA对酪氨酸酶(Tyr)作为对照(图2)A.B3-tubulin(与TUJ1染色)是神经元的标记物,在NSCs中没有表达,而围绕中央管道的SOX2-TUJ1-细胞被认为是含有Sox2缺失的细胞。通过CAS9-Sox2-gRNA电穿孔(图2B),定量显示Sox2在大量N...

Watch this Scientific Journal Video about Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes at JoVE.com

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

通过CAS9蛋白-gRNA复合物电穿孔直接基因敲除Axolotl脊髓神经干细胞

这里介绍的一个协议,通过将CAS9-gRNA复合物注射到脊髓中央管中,然后进行电穿孔,在axolotl脊髓中执行时间和空间限制基因敲除。

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

该协议允许快速和高度穿透时间和空间限制基因敲出轴脊髓神经干细胞的研究脊髓再生的分子机制。该技术允许研究神经干细胞中的基因功能，其医学再生，而不会被其他细胞类型的发育效应或效应所迷惑。Axolotl神经干细胞在很大程度上有助于它们再生脊髓的能力。了解再生机制可以带来对开发人类脊髓损伤治疗的见解。这种方法提供了对轴托洛神经干细胞如何对脊髓损伤进行再生反应的洞察。正确定位注射毛细管到脊髓中央柱可能具有挑战性，在测试动物身上练习有助于掌握该技术。查看管理可以显示如何执行注射，以及成功的注射是什么样子。为了准备Cas9导导RNA核蛋白混合物，混合5微克的Cas9核定位序列蛋白、4微克导导RNA和0.9微升10X Cas9缓冲液。然后将总体积与无核酶水一起带到 10 微升，如果不立即使用，则将 Cas9 导导 RNA 核蛋白混合物储存在零下 80 摄氏度。对于阿加罗斯电穿孔板制备，在杜尔贝科的PBS中制备200毫升2%的加糖，并在微波炉中溶解混合物。稍稍冷却后，将液体气糖倒入10厘米培养皿中，深度约为10毫米，让板在室温下凝固。当阿加罗斯是坚定的触摸，使用手术手术刀切开一个缝在每道菜的阿加罗斯保持尾巴直，一口井，以适应动物的身体，和两个额外的井，以举行电极。然后将盘子放在冰上，用冰冷的 DPBS 填充到边缘。要配置电穿孔器，请将孔隙脉冲设置为一个双极脉冲，持续时间为 70 伏，持续时间为 5 毫秒，间隔为 50 毫秒，且无电压衰减。将传输脉冲设置为四个 40 伏的双极脉冲，持续时间为 50 毫秒，间隔为 999 毫秒，电压衰减为 10%。然后将电极连接到电穿孔器并调整钳子，使它们相距七毫米，然后再将电极淹没在 PBS 的烧杯中。要准备玻璃微喷射毛细管，请根据制造商的说明对微管拉拔器执行斜坡测试，以确定热值。然后用指示参数拉取带锥形末端的喷射毛细管。拉后，使用立体显微镜和细钳以一定角度打破毛细管，使毛细管在尖端足够薄的位置倾斜，以瞄准轴椎的中央运河，同时保持足够强，足以刺穿皮肤。在加载尖端之前，以 1 比 30 的比例向核核蛋白混合物添加快速绿色 FCF 溶液，并使用凝胶加载移液器尖端将注射混合物的大约 5 微升加载到毛细管中。然后将毛细管安装在气动泵上，倾斜的尖端朝下。要配置气动泵，请将保持设置为每平方英寸 0.5 磅，将弹射设置为每平方英寸保持 2 磅，将持续时间设置为门控。要测试毛细管和泵设置，请将毛细管浸入一滴水中，然后踩脚踏板进行注射。注射混合物应该在缓慢但稳定的流中出来。要注入核蛋白混合物，请翻转要倒置在水中的斧头，以确认其反应，并使用环钳将一只动物转移到硅胶床上，动物的左侧朝上，尾巴指向右侧。将注射部位置于立体显微镜视野的中间，并调整微操纵器，使毛细管从视场右侧以 60 度角进入动物。识别脊髓和中央管后，缓慢地将毛细管移向脊髓中央管，直到它轻轻地刺穿皮肤和肌肉。当毛细管就位时，按住脚踏板将混合物注入中央运河。如果毛细管被正确放置，核核蛋白混合物将沿着中央运河以蓝线向两个方向扩散。正确定位毛细管可能具有挑战性。如果混合物未正确扩散，请按小步骤调整毛细管位置，同时保持脚垫。继续按住脚踏板，直到混合物到达脊髓和大脑心室的端囊。注射过程中可能需要调整喷射压力。注射后，立即使用环钳将动物转移到冰上准备好的气糖电穿孔板上，将尾巴放在缝隙内，以便被加糖夹。将电极放入尾部两侧的孔中，注意整个动物和电极被冰冷的 PBS 覆盖，然后按脚开关开始电穿孔。然后将动物返回水中进行监测，直到完全恢复，并以这种方式注入和电毒随后的动物。为了在蛋白质水平上评估淘汰的效率，获得尾部的横截面，并按照标准方案进行免疫组织化学分析。或者，提取电穿孔脊髓，并提取样品以获得DNA。然后执行基因分型PCR，然后进行桑格测序或下一代测序，以评估编辑的遗传位位百分比。Cas9 引导 RNA 复合物对 Sox2 的注射和电穿孔导致大多数脊髓神经干细胞中 Sox2 免疫反应性大量丧失，相比之下，Cas9 引导 RNA 复合物的注射和电穿孔对酪氨酸酶进行控制。在脊髓组织部分样本中对Sox2阳性细胞进行定量分析，发现Cas9 Sox2中Sox2表达细胞的数量显著减少，引导RNA电穿孔敲出动物，表明这种方法导致脊髓神经干细胞中高效且高穿透性基因敲出。在同时注射和电穿孔两个Cas9引导RNA复合物对Sox2和GGFP到转基因阿索洛茨与无处不在的GP表达，观察到高百分比的双敲出神经干细胞，表明该协议可用于灵活敲出多个基因。练习用毛细管定位中央运河，并放入配方与故障排除步骤，当注射不能正常工作时，这很有帮助。这项技术使研究人员能够研究神经干细胞中特有的再生基因的功能。操纵毛细管时小心，确保不要刺伤自己的手指。

Research

JoVE Journal

Genetics

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

科研

遗传学

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

利用 niper-cas9 在不损失目标活动的情况下通过定向进化, 最大限度地减少 crispr-cas9 的非目标效应

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...