Name: laser-assisted lentiviral gene delivery to mouse fertilized eggs
Uploaded: 2018-11-01T14:30:00
Description: Watch this Scientific Journal Video about Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs at JoVE.com

这项研究得到了国家卫生研究所 (nih)、国家环境卫生科学研究所 (niehs) 内部研究方案的支持。我们感谢罗伯特·彼得罗维奇博士和杰森·威廉姆斯博士对手稿的批判性解读和有益的建议。我们还要感谢贝恩德·格洛斯博士、淘汰赛核心公司、流式细胞术设施、荧光显微镜和成像中心以及 niehs 比较医学处的设施所作的技术贡献。我们要感谢比较医学处的 david goulding 先生和 nihs 成像中心的 lois wyrick 女士为我们提供了照片和插图。

该议定书中使用的所有动物程序和治疗方法都符合 nih/niehs 动物护理准则, 并得到 NIH/NIEHS 动物护理和使用委员会 (acuc) 的批准, 《2010-0004 动物议定书》。
1. 准备工作
<ol><li>购买具有兴趣基因的重组非繁殖慢病毒。本研究采用 lentivirus SBI511 表达科普足绿色荧光蛋白 (copgfp; 简称 gfp) 的伸长因子1a 启动子进行了透射。标准协议 2用于在每毫升 (tu/ml) 的滴度高于1e8...

<ol>
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慢病毒整合到宿主基因组中的能力使它们成为稳定基因传递的理想载体。慢病毒载体最多可以携带8.5 千卡的遗传物质, 这些遗传物质可以容纳特定细胞或诱导的启动子、选择标记或荧光分子。包含的基因组材料可以作为其宿主基因组的一部分进行复制, 并在所需的时间点进行调节以表达或停用。这些载体允许在不同的发展阶段对基因表达进行时空控制, 并将 lentiviruses 品牌作为基因传递的有力工具?...

该方法为小鼠受精卵透明带的渗透性提供了详细的指导, 使胚胎细胞可通过慢病毒进行基因传递。lent主义病毒是自然为有效地将基因传递给哺乳动物细胞而设计的。它们感染分裂和非分裂细胞, 并将慢病毒基因组整合到它们的宿主染色体1中。由于 vsv-g 蛋白2的广泛取向, 通过假型重组慢病毒与泡状口腔炎病毒糖蛋白 (vsv-g) 的分析, 使慢病毒宿主细胞的范围易于扩大。在转导之后, 慢病毒基因被稳定地整合和表达, 作为其宿主染色体的一部分, 创造了一个理想的工具, 产生转基因动物。如果传递给早期胚胎细胞, 慢病毒基因组被复制并在整个生物体中表达。慢病毒转导导致小鼠、大鼠、鸡、鹌鹑和猪3、4、5、6、7种转基因生物的产生。然而, 典型的慢病毒基因传递方法需要熟练的技术人员和专门的设备来克服包裹早期胚胎的透明带屏障。这种方法的总体目标是描述如何渗透带状疱疹使用激光, 以促进慢病毒基因的传递。
哺乳动物卵被透明带包围, 在受精后使其变硬, 以保护受精卵免受多精子的侵害, 并限制环境相互作用8,9。带形成了一个屏障, 使小病毒远离胚胎细胞, 直到胚胎孵化成囊胚。培养的小鼠受精卵在4天后孵化, 必须在孵化前植入假性小鼠体内, 才能正常发育到幼崽体内。因此, 对于转导, 慢病毒在孵化前从带状疱疹进入玻璃体周围腔, 即带状疱疹和胚胎细胞之间的空间。
透明带经常被去除用于人体卵子的体外受精, 以提高受精率10。然而, 清除小鼠透明带的化学作用对小鼠胚胎发育产生不利影响, 对胚胎细胞11,12 是有害的。其他将基因传递给小鼠受精卵的方法通过直接向细胞核13注入 dna 来克服透明带屏障.核微注射是将基因传递给胚胎的有效手段。然而, 由于每个胚胎都是单独固定的微注射, 这种做法对新手来说可能是费力和耗时的。
其他方法, 如电穿孔和光着色是有用的瞬态和短期基因传递到小鼠受精卵14,15,16。这些方法被广泛用于提供 crispr-cas9 组件和重组。然而, 基因的电穿孔和光化传递不能有效地用于创造转基因。从被刺破的小鼠附附虫中采集的精子也可以被慢病毒诱导, 并用于体外受精, 以产生转基因动物17,18,19, 20岁。
在该协议中, 利用激光渗透带的光病毒基因传递到小鼠胚胎。开发了 xyclone 激光, 用于体外受精21和胚胎干细胞22的培养。它是一个小的设备, 设置简单, 易于使用。一旦安装在显微镜上, 它就占据了物镜的空间, 随附的软件允许在透过显微镜目镜观察的同时瞄准激光 (见协议: 第3节)。一旦带状疱疹被 xyclone 激光射孔, 就可以将慢病毒引入基因传递的培养基23。多种慢病毒可用于同时传递多个基因的染色体整合。
该协议将描述如何分离和培养小鼠受精卵, 说明激光用于透明带穿孔, 并演示小鼠胚胎细胞的转导由慢病毒。

<table>
	<tbody>
		<tr>
			<td>CD510B-1 plasmid</td>
			<td>System Biosciences</td>
			<td>CD510B-1</td>
			<td>used to package the lentivirus expressing EF1a-copGFP</td>
		</tr>
		<tr>
			<td>Dimethylpolysiloxane</td>
			<td>Sigma</td>
			<td>DMPS5X</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td>used to remove cumulus cells</td>
		</tr>
		<tr>
			<td>XYClone Laser</td>
			<td>Hamilton Thorne Biosciences</td>
			<td></td>
			<td>perforating mouse fertilized eggs</td>
		</tr>
		<tr>
			<td>Non-Surgical Embryo Transfer (NSET) Device</td>
			<td>ParaTechs</td>
			<td>60010</td>
			<td>NSET of embryos</td>
		</tr>
		<tr>
			<td>KSOM medium</td>
			<td>Millipore</td>
			<td>MR-020P-5F</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>Composition of KSOM:</td>
			<td></td>
			<td></td>
			<td>mg/100mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>555</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>18.5</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>4.75</td>
		</tr>
		<tr>
			<td>MgSO4 7H2O</td>
			<td></td>
			<td></td>
			<td>4.95</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td></td>
			<td></td>
			<td>25</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td></td>
			<td>210</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>Na-Pyruvate</td>
			<td></td>
			<td></td>
			<td>2.2</td>
		</tr>
		<tr>
			<td>DL-Lactic Acid, sodium salt</td>
			<td></td>
			<td></td>
			<td>0.174mL</td>
		</tr>
		<tr>
			<td>10mM EDTA</td>
			<td></td>
			<td></td>
			<td>100&#181;L</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td></td>
			<td></td>
			<td>6.3</td>
		</tr>
		<tr>
			<td>0.5% phenol red</td>
			<td></td>
			<td></td>
			<td>0.1mL</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td></td>
			<td></td>
			<td>14.6</td>
		</tr>
		<tr>
			<td>MEM Essential Amino Acids</td>
			<td></td>
			<td></td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>MEM Non-essential AA</td>
			<td></td>
			<td></td>
			<td>0.5mL</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Millipore</td>
			<td>MR-015-D</td>
			<td>culturing embryos</td>
		</tr>
		<tr>
			<td>Composition of M2:</td>
			<td></td>
			<td></td>
			<td>mg/100mL</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td></td>
			<td></td>
			<td>25.1</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (anhydrous)</td>
			<td></td>
			<td></td>
			<td>16.5</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td></td>
			<td></td>
			<td>35.6</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Monobasic</td>
			<td></td>
			<td></td>
			<td>16.2</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td></td>
			<td></td>
			<td>35</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td></td>
			<td></td>
			<td>553.2</td>
		</tr>
		<tr>
			<td>Albumin, Bovine Fraction</td>
			<td></td>
			<td></td>
			<td>400</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>Na-HEPES</td>
			<td></td>
			<td></td>
			<td>54.3</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td></td>
			<td></td>
			<td>1.1</td>
		</tr>
		<tr>
			<td>Pyruvic Acid</td>
			<td></td>
			<td></td>
			<td>3.6</td>
		</tr>
		<tr>
			<td>DL-Lactic Acid</td>
			<td></td>
			<td></td>
			<td>295</td>
		</tr>
	</tbody>
</table>

laser-assisted lentiviral gene delivery to mouse fertilized eggs

慢病毒是将基因传递给哺乳动物细胞的有效载体。继转导之后, 慢病毒基因组被稳定地纳入宿主染色体, 并传递给后代。因此, 它们是创造稳定细胞系、体内传递指标和单个细胞受精卵的理想载体, 以创造转基因动物。然而, 小鼠受精卵和早期胚胎是由透明带保护的, 透明带是一种糖蛋白基质, 形成了抵御慢病毒基因传递的屏障。慢病毒太大, 无法穿透带状疱疹, 通常通过微注射病毒颗粒进入卵细胞周围腔, 即带状疱疹和胚胎细胞之间的空间。对高技能技术人员和专用设备的需求已最大限度地减少了小鼠胚胎基因传递慢病毒的使用。本文介绍了一种用激光射孔小鼠受精卵的渗透方案。激光穿孔不会对胚胎造成任何损害, 并允许慢病毒进入胚胎细胞进行基因传递。转基因胚胎可以在体外发育为囊胚, 如果植入假性小鼠体内, 就会发育成转基因幼崽。该协议中使用的激光是有效的, 易于使用。慢病毒提供的基因稳定地纳入小鼠胚胎细胞, 并可细菌传播。这是一种创造转基因小鼠的替代方法, 不需要微操作和微注射受精卵。

每天可在显微镜下检查分离/转染小鼠受精卵的发育情况 (图 1)。健康的胚胎在3-4天内发育成囊胚。在该协议中, 60-70% 未经处理的胚胎发育成囊胚 23.在114个激光穿孔的经导胚胎中, 54个发育为囊胚 (率为 47%), 46个囊胚表达 gfp (46/54 = 85%)23。
小鼠胚胎在收获当天被激光穿孔?...

Watch this Scientific Journal Video about Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs at JoVE.com

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

小鼠受精卵和早期胚胎受透明带保护, 透明带是一种糖蛋白基质, 形成了对抗基因传递的屏障。本文介绍了一种用激光射孔带带的协议, 用慢病毒载体传递胚胎细胞, 并创造转基因小鼠。

激光辅助慢病毒基因传递到小鼠受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

这是一种替代且易于使用的方法，通过激光穿孔的佐纳佩普伦西达和扁病毒基因传递，将感兴趣的基因传递到受精小鼠卵子。小鼠供体程序的激光辅助穿孔也适用于其他物种的受精卵，以便利其他类型的病毒或转染试剂的进入。这项技术的主要优点是，它不需要专门的技能，为受精小鼠卵子的微操作和微注射。展示受精小鼠卵子分离程序将是佩奇迈尔斯，一个研究科学家从比较医学分支在NIEHS。交配后16至24小时，根据标准协议，将卵巢和卵巢与阴道塞的雌性小鼠分离。收集所有组织后，将每个卵巢的安培分开，释放受精卵，周围是积木细胞，将细胞转移到一个35毫米的培养皿中，在新鲜的M2培养基中含有一个x hyaluronidase，在室温下孵育3至5分钟。移液几个次的鸡蛋释放的细胞，并转移鸡蛋到一个新的35毫米的菜含有M2培养，用移液洗掉酶。接下来，将洗净的鸡蛋转移到一个35毫米的盘中，含有钾，简单优化的介质，或KSOM，和移液器洗掉剩余的M2介质和透明酶。然后，将鸡蛋转移到35毫米组织培养处理板，用50微升的KSOM和两毫升二甲基二氧化硅烷的种子在37摄氏度、5%的二氧化碳和氧气以及90%的氮气下平衡两小时。当鸡蛋在孵化器中时，根据制造商的建议，设置并校准XYClone激光器。当卵子准备好时，将第一个滴盘放在激光显微镜阶段，然后手动聚焦佐纳佩鲁西达。在确认激光 LED 光通过目标可见后，移动显微镜阶段以 LED 激光瞄准 Zona，并使用计算机软件将 XYClone 激光设置为 250 微秒。将 LED 灯尺寸调整到适当的尺寸，用激光打孔三次，产生三个 10 微米的孔。快速进行穿孔至关重要，但请小心，因为受精卵不能保持在孵化器之外超过 15 分钟。然后，将穿孔的鸡蛋返回孵化器再两个小时。在第二次孵育结束时，用两个微升浓缩扁家病毒的微升处理50微升KSOM滴，无需混合。把鸡蛋还到孵化器四天。当卵子发育成胚胎时，使用非手术胚胎移植装置，将大约10-15个胚胎沉积在大约两个微升的KSOM中。胚胎移植后17天，允许怀孕小鼠自然分娩或必要时通过剖腹产收集新生儿。然后，从幼崽中收集组织进行基因分型，以确定转生成率。隔离和转导小鼠受精卵的发展可以每天在显微镜下检查。60-70%的健康、未经治疗的胚胎在三到四天内发育成胚胎。虽然大约47%的激光穿孔，转导胚胎发展成胚子，其中85%表示转导的基因感兴趣。瞄准激光变薄的佐纳，而不是创造一个洞，使发育中的胚胎受益于一个完好无损的，封装的佐纳佩鲁西达，同时保持宽松的扁病毒转导。与重组的扁病毒的转导，从伸长因子1A促进剂表达应付GFP诱导多个扁病毒整合，在蓝色LED灯下GFP的高表达。在尝试此程序时，重要的是要记住，虽然扁病毒整合到宿主基因组的能力使它们成为稳定基因传递的有力工具，但随机整合也可能引入插入性突变。这项技术可以使遗传学领域的研究人员迅速开发转基因动物模型，用于研究体内蛋白质生产或功能基因研究的疾病。按照这个程序，其他方法，如免疫组织化学可以执行从转导幼崽的分离组织样本，以回答有关位置和基因表达在各种感兴趣的组织的程度的其他问题。不要忘记，使用扁病毒可能是极其危险的。因此，请遵循您的机构指南，使用扁病毒。在处置前，请穿个人防护服，净化所有废物。

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SCIENTISTS IN ACTION

Windowing Chicken Eggs for Developmental Studies

发育研究的窗口鸡蛋

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

科研

生物学

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...