高效且具有成本效益的电穿孔方法研究颗粒细胞前体中的原代纤毛依赖性信号通路
Summary
在这里，我们提出了一种可重复的 体外 电穿孔方案，用于原代小脑颗粒细胞前体（GCP）的遗传操作，具有成本效益，高效且可行。此外，该协议还展示了一种简单的方法，用于原代GCP细胞中原代纤毛依赖性刺猬信号通路的分子研究。
Abstract
原代纤毛是一种关键的信号细胞器，几乎存在于从细胞表面转导刺猬（Hh）信号刺激的每个细胞上。在颗粒细胞前体（GCP）中，原代纤毛充当关键信号中心，通过调节Hh信号通路来协调前体细胞增殖。通过对通路成分的 体外 遗传操作来促进原发性纤毛依赖性Hh信号传导机制的研究，以可视化它们对原发性纤毛的动态定位。然而，使用目前已知的电穿孔方法在GCP的初级培养物中转染转基因通常成本高昂，并且通常导致细胞活力低和不良转染效率。本文介绍了一种高效、经济且简单的电穿孔方案，该方案显示出~80-90%的高转染效率和最佳的细胞活力。这是一种简单、可重复且高效的遗传修饰方法，适用于研究原代 GCP 培养物中原发性纤毛依赖性刺猬信号通路。
Introduction
小脑GCP由于其在体内对Hh信号通路的高丰度和高敏感性，被广泛用于研究神经元祖细胞类型中Hh信号通路的机制1，2，3，4。在GCP中，原发纤毛充当关键的Hh信号转导中枢5，协调前体细胞的增殖6，7，8。原发性纤毛上Hh信号传导成分的体外可视化通常具有挑战性，因为它们的内源性基础水平较低。因此，蛋白质表达水平的转基因修饰和目标基因的荧光团标记是在分子分辨率下研究该途径的有用方法。然而，使用基于脂质体的转染方法对 GCP 原代培养物进行遗传操作通常会导致转染效率低下，从而阻碍进一步的分子研究9。电穿孔可提高效率，但通常需要过多的供应商专用和电池类型受限的电穿孔试剂10。
本文介绍了一种高效且具有成本效益的电穿孔方法，用于操纵基仕伯原代培养物中的Hh信号通路组分。使用这种改良的电穿孔方案，绿色荧光蛋白（GFP）标记的平滑转基因（pEGFP-Smo）有效地递送到GCP，并实现了高细胞存活率和转染率（80-90%）。此外，如免疫细胞化学染色所证明的那样，转染的GCP对平滑激动剂诱导的Hh信号通路激活表现出高度敏感性，方法是将EGFP-Smo转运到原代纤毛。该协议应直接适用于涉及难以转染的细胞类型的 体外 遗传修饰的实验，例如人类和啮齿动物原代细胞培养物，以及人类诱导多能干细胞。
Protocol
Representative Results
Discussion
通过电穿孔法转染原代GCP培养物中的转染基因通常与细胞活力低和转染效率差有关9，10。本文介绍了一种具有成本效益且可重复的电穿孔方案，该协议已被证明具有高效率和可行性。此外，我们还展示了一种研究原代GCP细胞中原代纤毛依赖性Hh信号通路的简单方法。
其他常见的电穿孔方法通常需要昂贵的细胞类型特异性电穿孔试剂?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究获浸会大学种子基金及2级启动资助（RG-SGT2/18-19/SCI/009）、研究资助局合作研究基金（CRF-C2103-20GF）资助予C.H.H. Hor。
Materials
| GCP Culture | |||
| B27 supplement | Life Technologies LTD | 17504044 | |
| Cell strainer, 70 µm | Corning | 352350 | |
| DNase I from bovine pancreas | Roche | 11284932001 | |
| Earle’s Balanced Salt Solution | Gibco, Life Technologies | 14155063 | |
| FBS, qualified | Thermo Scientific | SH30028.02 | |
| GlutamMAXTM-I ,100x | Gibco, Life Technologies | 35050061 | L-glutamine substitute |
| L-cysteine | Sigma Aldrich | C7352 | |
| Matrigel | BD Biosciences | 354277 | Basement membrane matrix |
| Neurobasal | Gibco, Life Technologies | 21103049 | |
| Papain,suspension | Worthington Biochemical Corporation | LS003126 | |
| Poly-D-lysine Hydrobromide | Sigma Aldrich | P6407 | |
| SAG | Cayman Chemical | 11914-1 | Smoothened agonist |
| IF staining | |||
| Bovine Serum Albumin | Sigma Aldrich | A7906 | |
| Paraformaldehyde | Sigma Aldrich | P6148 | |
| Triton X-100 | Sigma Aldrich | X100 | |
| Primary antibody mix | |||
| Anti-GFP-goat ab | Rockland | 600-101-215 | Dilution Factor: 1 : 1000 |
| Anti-Arl13b mouse monoclonal ab | NeuroMab | 75-287 | Dilution Factor: 1 : 1000 |
| Anti-Pax6 rabbit polyclonal ab | Covance | PRB-278P | Dilution Factor: 1 : 1000 |
| Secondary antibody mix | |||
| Alexa Fluor 488 donkey anti-goat IgG | Invitrogen | A-11055 | Dilution Factor: 1 : 1000 |
| Alexa Fluor 555 donkey anti-mouse IgG | Invitrogen | A-31570 | Dilution Factor: 1 : 1000 |
| Alexa Fluor 647 donkey anti-rabbit IgG | Invitrogen | A-31573 | Dilution Factor: 1 : 1000 |
| DAPI | Thermo Scientific | 62247 | Dilution Factor: 1 : 1000 |
| Electroporation | |||
| CU 500 cuvette chamber | Nepagene | CU500 | |
| EPA Electroporation cuvette (2 mm gap) | Nepagene | EC-002 | |
| Opti-MEM | Life Technologies LTD | 31985070 | reduced-serum medium for transfection |
| pEGFP-mSmo | Addgene | 25395 | |
| Super Electroporator NEPA21 TYPE II In Vitro and In Vivo Electroporation | Nepagene | NEPA21 | electroporator |
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