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LB Agar, powder (Lennox L agar)

LB琼脂,粉末(Lennox L琼脂)

Company: Thermo Fisher Scientific
Catalog#: 22700025
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Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
Author:
Date:
2018-04-05
[Abstract]  For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones. [摘要]  对于疾病和基础科学研究而言,功能丧失(LOF)突变是非常重要的。 在这里,我们提供了一个简单的流线化协议来产生LOF iPSC系列,通过将CRISPR载体与附加型重编程质粒同时引入成纤维细胞,规避了传统基因编辑和已建立的iPSC系的克隆的技术挑战。 我们的实验已经产生了常染色体基因中所有3种基因型的几乎偶数。 此外,我们提供了一个详细的方法来维护和iPSC克隆的96孔板的基因分型。

【背景】CRISPR / Cas9技术允许简单且特异地针对特定基因组位置进行基因编辑。将该技术与诱导性多能干细胞(iPSC)的疾病建模和再生医学潜力相结合将继续对生物医学研究产生前所未有的影响。然而,使CRISPR / Cas9系统适应iPSC已经提出了几个挑战。在细胞系中进行基因编辑的传统方法是用表达Cas9蛋白质的质粒和指导RNA(gRNA)转染细胞,然后产生单克隆并筛选所需的遗传改变。不幸的是,iPSC不适用于单细胞克隆。已经开发了几种补充媒介和克隆方法来克服这一困难,但仍然充满昂贵的设备(低氧培养箱),困难的技术步骤(FACS分选的单个iPSC的存活)或劳动密集型方案(亚克隆)(Forsyth ,2006; Miyaoka ...

Induction of Connexin-hemichannel Opening
Author:
Date:
2014-09-05
[Abstract]  Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes and immune cells. Hemichannels allow the passage of molecules of up to 1-2 kDa along the concentration gradient. When surface-exposed, hemichannels mediate the exchange of molecules between the cytosol and the extracellular space. Hemichannels are closed by default, but several cues inducing their opening have been described, e.g. a drop in the extracellular Ca2+ concentration (Evans et al., 2006) or infection with enteric pathogens (Puhar et al. ... [摘要]  连接蛋白(Cxs)是脊椎动物的内在膜蛋白,其结合形成六聚体跨膜通道,称为半通道。已经描述了21种Cx类型,其根据它们的分子量命名。 Cxs在许多细胞类型中表达,例如上皮细胞,星形胶质细胞和免疫细胞。半通道允许高达1-2kDa的分子沿浓度梯度通过。当表面暴露时,半通道介导细胞溶质和细胞外空间之间的分子交换。半通道被默认关闭,但是已经描述了诱导其开放的几个线索,例如,细胞外Ca 2+浓度的下降(​​Evans等人, 2006);或用肠道病原体感染(Puhar et al。,2013; Tran Van Nhieu等人,2003)。该方案与上皮细胞,特别是与极化和非极化肠上皮TC7细胞和用Cx26或Cx43稳定转染的Hela细胞(Paemeleire等人,2000)一起使用。然而,它可能可用于其他Cx表达细胞类型。是否半通道是开放的可以通过电生理学或通过测量半通道可渗透分子(例如,ATP)在细胞外介质中的释放或半通道可渗透的质膜不渗透分子的摄取来确定[例如,荧光染料溴化乙锭 - 参见相关协议"通过Connexin Hemichannels进行染料吸收实验"(Puhar和Sansonetti,2014 )]。

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