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Penicillin-Streptomycin (5,000 U/mL)

青霉素 - 链霉素,液体

Company: Thermo Fisher Scientific
Catalog#: 15070063
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Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection
Author:
Date:
2018-09-05
[Abstract]  Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) stereotaxic injection of the lentivirus into Area X of juvenile zebra finches; 4) analysis of song learning and song performance in juvenile and adult zebra finches. These methods complement the methods employed in recent works that showed changing FoxP2 gene expression in Area X with lentivirus or adeno-associated virus leads to impairments in song behavior. [摘要]  在这里,我们提供了一个详细的逐步协议,用于使用慢病毒操纵幼年斑胸草雀X区的miRNA表达,并分析对歌曲学习和歌曲表现的影响。 该方案有四个部分:1)使慢病毒构建体过表达miRNA miR-9; 2)包装慢病毒载体; 3)将慢病毒立体定位注入少年斑胸草雀X区; 4)青少年和成年斑马雀的歌曲学习和歌曲表演分析。 这些方法补充了近期工作中使用的方法,这些方法显示,在区域X中用慢病毒或腺相关病毒改变 FoxP2 基因表达导致歌曲行为的损害。

【背景】具有良好特征的歌曲行为和基础神经回路的斑胸草雀提供了独特的动物模型来研究声音通信和相关感觉 - 运动学习的神经机制。近年来,一些实验室开始使用病毒载体来操纵斑胸草雀脑中的基因表达并研究其功能后果。这些努力通过对 FoxP2 基因的研究得到了最好的说明,该基因编码叉头盒p2转录因子。 FoxP2蛋白控制着数百个在神经系统发育中起重要作用的下游基因的表达。人类 FoxP2 基因的突变导致言语和语言障碍(Lai et al。,2001)。在鸣禽中,斑马雀X区域的 FoxP2 基因的敲除或过表达,对于声乐学习至关重要的基底神经节核,严重损害了歌曲的行为(Haesler et al。, 2007; Murugan et al。,2013; Heston and ...

3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
Author:
Date:
2018-06-05
[Abstract]  Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, ... [摘要]  细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而,在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用,并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性(Edmondson等人,2014)。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型(肿瘤球体)在该领域已经获得了越来越多的普及(Weiswald等人,2015)。使用基质嵌入或封装的球体,悬滴培养的球体,磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中,我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样,该方案也可用于直接从肿瘤组织中分离3D癌细胞系。

【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法(Pampaloni等人,2007; ...

In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
Author:
Date:
2018-05-20
[Abstract]  Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung. [摘要]  早期鼠肺发育,包括原基的规范,早期内胚层的构图和区域祖细胞命运的确定,受到严格的调控。 培养移植胚胎肺组织的能力提供了一种易处理的模型来研究调节这些过程的细胞相互作用和旁分泌因子。 我们提供最新的协议,以建立这种文化模式及其应用来研究肺部发育中的刺猬信号。

【背景】小鼠肺发育起始于前肠前内胚层的内胚层憩室(E9.5),随后关闭近端气管食管中隔以形成不同的气管和食道管(Minoo和King,1994)。原始内胚层管的随后分支通过E12.5产生平面肺结构,随后正交分支产生成熟肺的三维结构特征(Metzger等人,2008)。在E12.5之前分离的肺遗传的平面结构适合于在空气液体界面进行体外培养(Carraro等人,2010; Del Moral和Warburton, 2010)。胚胎肺通过解剖使用立体显微镜在亮场照明下或通过与谱系追踪和荧光报道分子偶联时的荧光照明进行分离。在这里,我们描述了使用Shh Cre / Rosa mTmG报告小鼠,其允许Cre介导的从约E8.75起在前部前肠内胚层内的膜定位的GFP的激活(Montgomery <等人,2007;高斯等人,2009; Yao等人,2017)。因此,肺内胚层通过红色荧光由绿色荧光和周围组织显现,从而允许清晰识别和显微切割包括肺在内的发育内胚层结构,并在体外培养期间成像。

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