TOPO^TM TA Cloning^TM Kit, Dual Promoter, without competent cells

TOPO ^® TA Cloning ^® Kit，Dual Promoter，无感受态细胞

公司名称: Thermo Fisher Scientific

产品编号: 450640

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Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap

Author:

Jonas Mechtersheimer, Stefan Reber and Marc-David Ruepp,

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 / ...

Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR

Author:

Bianca Hoffmann and Bastian Grewe,

Date:

2013-04-20

[Abstract] Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation ... [摘要] 病毒颗粒的形成和基因组逆转录病毒RNA包装到这些颗粒中是病毒复制周期晚期的重要步骤。可以使用定量逆转录酶-PCR（RT-qPCR）为基础的方法研究将病毒或细胞RNA掺入病毒颗粒中的效率。在从感染的或转染的细胞分离细胞质RNA并提取病毒颗粒相关的RNA后，测定两种级分中存在的特异性RNA水平。病毒体相关的和细胞质RNA的比率定义了衣壳化效率（Brandt等人，2007; Blissenbach等人，2010; Grewe等人，/em>，2012）。

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TOPOTM TA CloningTM Kit, Dual Promoter, without competent cells

TOPO ® TA Cloning ® Kit，Dual Promoter，无感受态细胞

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TOPO^TM TA Cloning^TM Kit, Dual Promoter, without competent cells

TOPO ^® TA Cloning ^® Kit，Dual Promoter，无感受态细胞