Evsen Lale, Doetzlhofer Angelika
The Solomon H. Snyder Department of Neuroscience, The Center for Sensory Biology, The Johns Hopkins University, School of Medicine.
The Solomon H. Snyder Department of Neuroscience, The Center for Sensory Biology, The Johns Hopkins University, School of Medicine;
J Vis Exp. 2016 Apr 17(110):53864. doi: 10.3791/53864.
Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2´-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.
鸡胚是研究胚胎发育的理想模型系统，因为可以相对轻松地在卵内进行基因功能操作。内耳听觉感觉器官对我们的听力至关重要。它包含一种高度特化的感觉上皮，由机械转导毛细胞（HCs）和周围的神经胶质样支持细胞（SCs）组成。尽管听觉器官在结构上存在差异，但调节听觉器官发育的分子机制在哺乳动物和鸟类之间具有进化保守性。卵内电穿孔在很大程度上仅限于E1 - E3的早期阶段。由于听觉器官在E5时发育相对较晚，在E3之后通过卵内电穿孔对听觉器官进行操作很困难，因为鸡胚在后期发育进程已推进。本文介绍的方法是一种通过卵内微电穿孔在发育中的鸡听觉感觉器官的E4 - E4.5阶段靶向感兴趣基因的瞬时基因转移方法。该方法适用于使用基于常规质粒DNA的表达载体进行功能获得和功能缺失研究，并且可以通过在电穿孔时向整个胚胎中添加EdU（5-乙炔基-2'-脱氧尿苷）与卵内细胞增殖测定相结合。使用绿色或红色荧光蛋白（GFP或RFP）表达质粒可使实验者快速确定电穿孔是否成功靶向发育中的内耳的听觉部分。在这篇方法论文中，展示了GFP电穿孔标本 的代表性实例；电穿孔后18 - 96小时收获胚胎，并通过RNA原位杂交确认GFP靶向听觉器官的前感觉区域。该方法论文还提供了使用胸苷类似物EdU分析细胞增殖的优化方案；提供了一个结合免疫标记和点击EdU化学的基于EdU的细胞增殖测定实例。
