| Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
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Author:
Date:
2017-09-05
[Abstract] Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).
[摘要] 心脏神经嵴细胞（CNCC）起源于神经管的背部边缘，位于第3个体节的耳穴和尾缘之间，并迁移到咽弓和心脏。 我们已经表明，纤连蛋白（Fn1）通过调节CNCCs到咽弓动脉周围的血管平滑肌细胞的分化，在CNCC的发展中起重要作用（Wang and Astrof，2016）。 该方案描述了CNCC与神经管和尾尾弓的分离，以及神经嵴衍生细胞分化成平滑肌细胞。 该方案从（Newgreen和Murphy，2000; Pfaltzgraff等人，2012）改编。
【背景】以前发表的方案描述了从神经管分离神经嵴细胞。然而，在耳孔和第三体细胞之间的神经管区域中的神经嵴细胞包括有助于许多不同细胞类型的神经嵴细胞群体;例如，迷走神经嵴细胞也来自该区域。在该方案中，我们修改了用于分离心脏神经嵴细胞的常规方法。而不是使用神经管，我们在胚胎期（E）9.5（22-25个体节期）使用尾部咽部弓形区。这是在将心脏神经嵴细胞分化为血管平滑肌细胞之前。神经嵴培养物通常含有污染性间充质细胞，通常表达平滑肌基因。为了鉴定神经嵴衍生细胞，我们从以下交叉产生的胚胎中分离出神经嵴细胞：Fn1flox / flox; ROSAmTmG / mTmG雌性小鼠×Fn1 +/-;Tfap2αIRESCre/ ...
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| Generation of Mouse Lung Epithelial Cells
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Author:
Date:
2013-08-05
[Abstract] Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+;p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following ...
[摘要] 尽管体内模型对于评估整个生物体生理学，病理学和对治疗的总体反应的各个方面是优异的，但是在哺乳动物模型系统中评估基本细胞功能和分子事件是具有挑战性的。因此，有利的是在精制和较便宜的设置中进行这些研究。一种方法涉及利用来自所评估的模型的细胞。产生这样的细胞的方法基于起始细胞和通常细胞的遗传学而变化。在这里，我们描述了从肺部产生上皮细胞的步骤 LSL-G12D/+ ; LSL-R172/+ 小鼠（Kasinski和Slack，2012）。这些小鼠在重组酶依赖性去除转基因中的终止盒并随后表达Kra -G12D 和后产生侵袭性肺腺癌> p53 R172 。虽然该方案可用于从其他遗传背景产生上皮细胞系，但应注意的是，Kras ;此处产生的p53 细胞系能够在培养物中增殖，而没有任何额外的遗传操作，其对于侵略性较低的背景通常是需要的。
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