Name: production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex
Uploaded: 2018-01-09T12:30:00
Description: Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

这份出版物的研究报告得到了国家卫生研究院的过敏和传染性疾病研究所 (NIAID) 的支持, 其奖项编号是 1R41OD021979 (对此) 和下一代 BioGreen 21 的研究补助金。计划, 大韩民国, 格兰特号PJ01107704。PJ01107703 (对 IK)。内容完全是作者的责任, 并不一定代表国家卫生研究院或 BioGreen 21 的官方观点。我们感谢 Dr. Nikolas Robl 编辑手稿。

本议定书所述程序由犹他州立大学动物保育和使用委员会 (IACUC) 批准 (IACUC 议定书: 2484)。本议定书使用的仓鼠是成人 (6-10 周龄) LVG 应变金黄叙利亚仓鼠。所有仓鼠都安置在犹他州立大学 Bioinnovation 中心的 vivarium。室温设置在23° c, 湿度设置为 40-50%, 光周期设置为 14 l:10 d (浅色: 深色)。只要有可能, 胚胎操作和 surugical 程序都应该用无菌技术进行。
1. sgRNA 和 Cas9/sgRNA 均一 (RNP) 的?...

<ol>
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A., 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=Syrian+hamster+as+a+permissive+immunocompetent+animal+model+for+the+study+of+oncolytic+adenovirus+vectors.">Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors.</a> Cancer Res. 66 (3), 1270-1276 (2006).</li><li>Thomas, M. A., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Syrian+hamster+as+an+animal+model+for+oncolytic+adenovirus+vectors.">Use of the Syrian hamster as an animal model for oncolytic adenovirus vectors.</a> Methods Mol Med. , 169-183 (2007).</li><li>Hogarth, C. A., Roy, A., Ebert, D. 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为了更好地利用黄金叙利亚仓鼠的潜力作为人类疾病的模型, 我们开发了一个 PN 注射协议, 以提供一个 CRISPR/Cas9 复合体为目标的仓鼠基因组。PN 注入协议优化了几个关键变量, 包括胚培养基、温度和光的波长13。还有几个仓鼠特定的动物处理程序, 需要遵循成功地进行基因打靶的仓鼠。例如, 性成熟的雌性仓鼠有稳定的4天的动情周期, 不能与外源激素同步 (例如PMSG)。因此, ?...

金黄叙利亚仓鼠 (Mesocricetus 鲫) 是生物医学研究中使用最广泛的啮齿动物之一。根据美国农业部的数据, 2015年美国使用了大约10万只仓鼠, 这代表了动物福利法 (http://www.aphis.usda.gov) 所涵盖的物种在实验室动物总数中的13%。2017年3月10日)。
在研究一些人类疾病时, 仓鼠比其他啮齿动物提供了一些优势。例如, nitrosobis (2-代丙基) 胺 (防喷) 诱导的胰胆管癌的组织病理学与人胰腺肿瘤相似, 而防喷治疗主要诱发大鼠甲状腺肿瘤和肺、肝肿瘤鼠标1。由于仓鼠是唯一发现支持复制腺的小鼠, 它们也是测试 adenovirus-based 溶载体和 anti-adenovirus 药物的选择模型2,3,4。另一个例子, 其中仓鼠模型提供了比老鼠和老鼠的优势是在高脂血症的研究。人和仓鼠在脂质代谢通路上具有很大的相似性, 两个物种携带胆固醇酯转运蛋白 (CETP) 的基因编码, 这在脂代谢中起着中心作用, 而 CETP 在小鼠和大鼠中则不存在5。此外, 在感染埃博拉病毒6后, 仓鼠发展出血性疾病更能代表人类的表现。仓鼠也是研究动脉粥样硬化的选择模型7, 口腔癌的8, 和炎症病9。最近, 它也已经证明, 仓鼠是高度敏感的安第斯山脉病毒感染和发展汉坦病毒性肺综合征样疾病, 提供了唯一的啮齿动物模型的安第斯山脉感染10。
为了解决新的基因动物模型无法满足的需要, 研究人类疾病, 没有可靠的小鼠模型, 我们最近成功地将 CRISPR/Cas9 系统应用到仓鼠, 并产生了几行基因工程仓鼠11。仓鼠受精卵对环境环境非常敏感, 因此在其他物种中开发的 PN 注射协议是不合适的。因此, 我们开发了一种用于仓鼠的 PN 注射协议, 它适用于处理仓鼠胚胎的特殊要求体外。在这里, 我们描述了详细的 PN 注射过程中使用的 CRISPR/Cas9 系统和相应的步骤, 从单一指南 RNA (sgRNA) 的准备转移到接受雌性注射胚胎。

<table>
	<tbody>
		<tr>
			<td>Cas9</td>
			<td>Invitrogen</td>
			<td>B25640</td>
			<td>1 ug/ul (~6.1 uM)</td>
		</tr>
		<tr>
			<td>GeneArtTM Precision Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Albumin from human serum</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td>For cultivation medium</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>NI-150</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>New Brunswick</td>
			<td>Galaxy 14S</td>
			<td>For embryo cultivation</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>PB-7</td>
			<td>For making injection needles</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti-S</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>invitrogen</td>
			<td>SMZ745T</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M1840</td>
			<td>Keep in dark</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4877-2000IU</td>
			<td>For superovulation</td>
		</tr>
	</tbody>
</table>

production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex

在小鼠体内首次建立了原 (PN) 注射技术, 将国外的遗传物质引入到单细胞阶段胚胎的核中。引入的遗传物质可以融入胚胎基因组, 并在移植后的胚胎转移到寄养母亲之后, 利用外来的遗传信息产生转基因动物。在小鼠成功后, PN 注射液已成功应用于其他许多动物品种。近年来, PN 注射成功地引入了基因修饰的试剂, 如 CRISPR/Cas9 系统, 在几个实验室和家畜物种中实现 site-specific 的基因修饰。除了掌握特殊的显微注射技术, 通过 PN 注射液生产转基因动物外, 研究人员还必须了解目标物种的繁殖生理和行为, 因为每个物种都呈现出独特挑战.例如, 金黄叙利亚仓鼠胚胎有独特的处理要求在体外, 因此 PN 注射技术是不可能的在这个种类直到最近突破由我们的小组。通过我们的物种改良 PN 注射协议, 我们成功地生产了几个基因敲除 (KO) 和敲 (基) 仓鼠, 已成功地用于人类疾病模型。在这里, 我们描述了 PN 注射程序, 交付 CRISPR/Cas9 复合物的受精卵的仓鼠, 胚胎处理条件, 胚胎移植程序, 和畜牧业所需的生产转基因仓鼠。

所述协议在生产转基因仓鼠中的效率取决于以下两个关键步骤的结果: 受赠雌性的活产率和预期基因修饰的活幼崽数。活产率是胚胎质量的直接结果, 是个体进行 PN 注射和胚胎移植过程的技术。为了确保纵胚胎的发育潜能不会受到损害 , 在对仓鼠胚胎进行的体外处理过程中需要非常小心。我们定期获得 60-80% 的 pseudopregnant 受赠女性的活产率。表 3演示了?...

Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

原 (PN) 注射的聚类定期 interspaced 短复发重复 (CRISPR) 和 CRISPR 相关 protein-9 核酸 (CRISPR/Cas9) 系统是一种高效的方法生产基因工程金黄叙利亚仓鼠。在此, 我们描述了详细的 PN 注射协议生产的基因剔除仓鼠与 CRISPR/Cas9 系统。

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

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