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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Agarose | Sigma-Aldrich | A9539 | |
| Benzocaine | Sigma-Aldrich | E1501-100G | |
| Benzocaine 0.03 % (wt/vol) | Mix 500 ml of 10× TBS, 500 ml of 400% (wt/vol) Holtfreter’s solution and 30 ml of 10% (wt/vol) benzocaine stock solution. Fill up the volume to 10 L with dH2O. The solution can be stored at room temperature for up to 6 months. | ||
| Benzocaine 10 % (wt/vol) | 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. | ||
| Borosilicate glass capillaries 1.2 mm O.D., 0.94 mm I.D. | Stutter Instrument | BF120-94-8 | |
| CaCl2·2H2O | Merck | 102382 | |
| CAS9 buffer, 10x | Mix 200 mM HEPES and 1.5 M KCl in RNase-free water. Adjust pH to 7.5. Filter sterilize, aliquot and store at −20 °C for up to 24 months | ||
| CAS9-NLS protein | PNA Bio | CP03 | |
| Cell culture dishes, 10cm | Falcon | 351029 | |
| Dumont #5 – Fine Forceps | Fine Scientific Instruments | 11254-20 | |
| Electroporator | Nepa Gene | NEPA21 | |
| BEX | Pulse Generator CUY21EDIT II | ||
| Fast Green FCF | Sigma-Aldrich | F7252-5G | |
| Fast Green FCF Solution, 5x | Dissolve 12.5 mg of Fast Green FCF powder in 10 mL of 1× PBS. | ||
| Flaming/Brown Micropipette Puller | Stutter Instrument | P-97 | |
| Holtfreter’s solution 400% (wt/vol) | Dissolve 11.125 g of MgSO4·7H2O, 5.36 g of CaCl2·2H2O, 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. | ||
| KCl | Merck | 104936 | |
| MgSO4·7H2O | Merck | 105886 | |
| Microloader pipette tips | Eppendorf | 5242956003 | |
| Micromanipulator | Narishige | MN-153 | |
| NaCl | Merck | 106404 | |
| Pneumatic PicoPump | World Precision Instruments | SYS-PV830 | |
| Ring Forceps | Fine Scientific Instruments | 11103-09 | |
| Stereomicroscope | Olympus | SZX10 | |
| Tris base | Sigma-Aldrich | T6066 | |
| Tris-buffered saline, 10x | 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. | ||
| Tweezers w/Variable Gap 2 Round Platinum Plate Electrode, 10mm diameter | Nepa Gene | CUY650P10 |
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