The combination of limb-bud removal and in ovo electroporation techniques: A new powerful method to study gene function in motoneurons undergoing lesion-induced cell death
The combination of limb-bud removal and in ovo electroporation techniques: A new powerful method to study gene function in motoneurons undergoing lesion-induced cell death
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DOI：
10.1016/j.jneumeth.2014.10.022
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发表时间：
2015-01
影响因子：
3
通讯作者：
Veronica La Padula;Sophie Koszinowski;K. Krieglstein
中科院分区：
文献类型：
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作者：
Veronica La Padula;Sophie Koszinowski;K. Krieglstein
鸡胚胎是发育生物学研究的重要模式生物。目前，从外科手术到分子生物学方法，已经建立了许多关于该模型的技术，以回答细胞生物学的重大问题。研究与运动神经元（MN）存活和细胞死亡有关的基因对于更好地理解导致MN退行性疾病（如肌萎缩侧索硬化症（ALS）和运动周围神经病）的分子机制至关重要。新方法在此，我们描述了一种众所周知的能够诱导MN细胞死亡的外科手术，即肢体芽切除术（LBR），与分子生物学中用于检测活生物体中基因功能的非常流行的方法卵电穿孔（IOE）。这项工作的目的是提供一种有效的方法，调查基因参与的MNs生存和细胞死亡的损伤conditions.ResultsOur方法允许成功的电穿孔的40-50%，LBR侧的MNs在手术后早期和晚期具有较高的生存率。与其他方法的比较与已经相比，这种改良的LBR技术结合IOE可以实现更高的MN表达效率结论我们的工作开启了在一个灵活和廉价的动物模型上以高重复性和高效率筛选参与MN存活或细胞死亡的大量基因的可能性。LBR/IOE技术为后续影响MNs存活的哺乳动物疾病模型的优化研究开辟了新的途径。
BackgroundThe chicken embryo is an important model organism for developmental biology studies. At present, many techniques on this model have been set up, from surgical procedures to molecular biology methods, to answer capital questions of cell biology. The study of the genes involved in motoneurons (MNs) survival and cell death is critical for a better understanding of the molecular mechanisms leading to MNs degenerative diseases, such as amyothophic lateral sclerosis (ALS) and motor peripheral neuropathies.New methodHere, we describe the combination of a well known surgical procedure able to induce MNs cell death, the limb-bud removal (LBR), with a very popular method used in molecular biology to test gene function in living organisms, thein ovoelectroporation (IOE). The aim of this work is to provide an effective method for the investigation of genes involved in MNs survival and cell death under lesion conditions.ResultsOur method allows the successful electroporation of the 40–50% of MNs on the side of LBR with a high survival rate early and late after procedure.Comparison with other methodsThis modified LBR technique combined with IOE allows a higher MN expression efficiency compared to an already published method.ConclusionsOur work opens the possibility of screening a multitude of genes involved in MNs survival or cell deathin vivowith high reproducibility and efficiency on a flexible and inexpensive animal model. The LBR/IOE technique opens a new way for the optimization of subsequent studies on mammalian models of diseases affecting MNs survival.