激光辅助慢病毒基因传递到小鼠受精卵
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| CD510B-1 plasmid | System Biosciences | CD510B-1 | used to package the lentivirus expressing EF1a-copGFP |
| Dimethylpolysiloxane | Sigma | DMPS5X | culturing embryos |
| hyaluronidase | Sigma | H3506 | used to remove cumulus cells |
| XYClone Laser | Hamilton Thorne Biosciences | perforating mouse fertilized eggs | |
| Non-Surgical Embryo Transfer (NSET) Device | ParaTechs | 60010 | NSET of embryos |
| KSOM medium | Millipore | MR-020P-5F | culturing embryos |
| Composition of KSOM: | mg/100mL | ||
| NaCl | 555 | ||
| KCl | 18.5 | ||
| KH2PO4 | 4.75 | ||
| MgSO4 7H2O | 4.95 | ||
| CaCl2 2H2O | 25 | ||
| NaHCO3 | 210 | ||
| Glucose | 3.6 | ||
| Na-Pyruvate | 2.2 | ||
| DL-Lactic Acid, sodium salt | 0.174mL | ||
| 10mM EDTA | 100µL | ||
| Streptomycin | 5 | ||
| Penicillin | 6.3 | ||
| 0.5% phenol red | 0.1mL | ||
| L-Glutamine | 14.6 | ||
| MEM Essential Amino Acids | 1mL | ||
| MEM Non-essential AA | 0.5mL | ||
| BSA | 100 | ||
| M2 medium | Millipore | MR-015-D | culturing embryos |
| Composition of M2: | mg/100mL | ||
| Calcium Chloride | 25.1 | ||
| Magnesium Sulfate (anhydrous) | 16.5 | ||
| Potassium Chloride | 35.6 | ||
| Potassium Phosphate, Monobasic | 16.2 | ||
| Sodium Bicarbonate | 35 | ||
| Sodium Chloride | 553.2 | ||
| Albumin, Bovine Fraction | 400 | ||
| D-Glucose | 100 | ||
| Na-HEPES | 54.3 | ||
| Phenol Red | 1.1 | ||
| Pyruvic Acid | 3.6 | ||
| DL-Lactic Acid | 295 |
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