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Gilson pipettes

Company: Scientific Laboratory Supplies
Catalog#: F123601
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Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
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Date:
2018-10-20
[Abstract]  This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap. [摘要]  该协议描述了CRISPR-Trap从设计基因靶向策略到验证成功编辑的克隆的应用,所述克隆在各种人细胞系上得到验证,其中人类诱导的多能干细胞(hiPSC)。 CRISPR-Trap优于常规方法的优点是从靶基因完全去除任何内源全长转录物。 CRISPR-Trap适用于在其第一个外显子中没有编码序列或编码序列很少的任何靶基因。 到目前为止,已经使用CRISPR-Trap成功编辑了几种人细胞系和不同基因。

【背景】CRISPR / Cas9技术的出现促进了基因敲除和基因编辑的基因组靶向。执行敲除的常规方法依赖于引入移码导致过早终止密码子(PTC),截短开放阅读框(ORF)以及随后通过无义介导的mRNA衰变(NMD)降解靶基因的转录物。 。这种方法的一个可能的缺陷是全长转录物,其可以逃避NMD并产生具有残余或甚至显性负功能的C末端截短蛋白。该协议提出了CRISPR-Trap,这是我们最近建立的一种方法(Reber et al。>,2018),成功编辑后将阻止从靶基因位点表达任何全长转录本(图1)。简而言之,这种方法针对CRISPR / ...

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
Author:
Date:
2018-10-05
[Abstract]  Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ... [摘要]  核糖核蛋白颗粒(mRNP)是由mRNA和RNA结合蛋白(RBP)组成的复合物,其控制mRNA转录定位,转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件,如poly(A)尾或5'帽结构,这通过应用广泛使用的方法如oligo(dT)纯化来阻止它们的鉴定。在这里,我们描述了基于现有的交联和免疫沉淀(CLIP)方法的修饰的交联亲和纯化方案(cCLAP),以分离mRNP中可被去腺苷酸化,去除和/或部分降解的mRNA,从而开启了检测不同的可能性。非编码RNA(ncRNA)的类型。分离后,将RNA进行衔接子连接,然后进行下一代测序(NGS)。由于快速有效的交联和淬灭步骤,该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体(PB)或应力颗粒(SG)。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀,然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法(Tagwerker et al。,2006; Hafner et al。,2010; Kishore et al。,2011)。 ...

Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates
Author:
Date:
2018-10-05
[Abstract]  Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein’s native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting. [摘要]  蛋白质聚集是几种神经退行性疾病的标志,并且与蛋白质体内平衡受损有关。 这种不平衡是由蛋白质天然构象的丧失引起的,最终导致其在细胞内聚集或异常定位。 使用 C。 线虫聚谷氨酰胺疾病模型,我们详细描述了过滤阻滞测定,一种捕获醋酸纤维素膜中蛋白质聚集体的方法,并允许通过免疫印迹进行检测和定量。
【背景】帕金森氏症,阿尔茨海默氏症和多聚谷氨酰胺疾病等神经退行性疾病的一个病理特征是在大脑不同区域存在蛋白质聚集物(Soto,2003; Stroo et al。,2017)。在多谷氨酰胺疾病的情况下,编码序列中谷氨酰胺(CAG)重复的异常扩增扰乱了蛋白质的天然折叠。结果,错误折叠的蛋白质暴露其氨基酸序列的区域,这使得它易于与其他蛋白质聚集,形成大的,不溶的聚集体,这可能妨碍正常的细胞功能(综述于Kuiper 等人 ,2017)。

已经开发了几种用于检测不溶性蛋白质聚集体的方法,包括例如染料结合测定(例如,硫磺素T,刚果红,NIAD-4)和电子显微镜检查。过滤阻滞测定是一种快速而灵敏的方法,可检测和定量体内和体外形成的蛋白质聚集体,包括聚谷氨酰胺(Scherzinger et al。 ,1997; Wanker et al。,1999),α-突触核蛋白(Recasens et al。,2018),和amyloid-beta聚集体(Bieschke et ...

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