| A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
|
|
Author:
Date:
2017-06-05
[Abstract] The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of ...
[摘要] 聚类规则间隔短回文重复（CRISPR）/ CRISPR相关蛋白9（Cas9）系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成：单导RNA（sgRNA）和Cas9。在特定靶序列引入双链断裂（DSB）需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的，因为它需要在体外转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA：CRISPR RNA（crRNA）和反式激活性crRNA（tracrRNA）进行靶向。这两种小RNA可以化学合成，这使得与sgRNA相比，这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而，已经开发了改进形式的Cas9以最小化离靶效应。例如，仅当两个引导RNA在规定的距离内结合相对的链时，切口酶型Cas9（nCas9）和FokI结构域融合的催化无活性的Cas9（FokI-dCas9; fCas9）才诱导DSB。在本协议中，我们描述了使用结合crRNA，tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂，而且可以降低脱靶效应的风险。
背景 ...
|
|
|
| RNA-dependent RNA Polymerase Assay for Hepatitis E Virus
|
|
Author:
Date:
2017-04-05
[Abstract] RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay the RdRp activity of many other viruses, this assay utilizes DIG-11-UTP as a nonradioactive alternative to 32P-UTP, thereby increasing the convenience of performing the assay.
[摘要] RNA依赖性RNA聚合酶（RdRp）对RNA病毒的病毒RNA的复制至关重要。 它合成病毒基因组RNA的互补链，其随后用作模板以产生更多的病毒基因组拷贝。 该测定法测定戊型肝炎病毒（HEV）RdRp的活性。 与可用于测定许多其他病毒的RdRp活性的方案相比，该测定法使用DIG-11-UTP作为32P-UTP的非放射性替代物，从而增加了进行测定的便利性。
没有测定可测量HEV RdRp的活性。 已经使用放射性标记的核苷酸（Behrens等人，1996）在少数其他病毒如丙型肝炎病毒中测量了RdRp活性。 我们已经调整了Behrens等人所描述的协议。 （1996），并将其修饰为建立非放射性测定方案，其依赖于将DIG-11-UTP掺入反义RNA链中作为HEV RdRp的活性的量度。 该测定使用体外合成的病毒RNA片段作为模板，以使用基于化学发光的策略来测量从人肝癌细胞纯化的HEV RdRp蛋白的活性。
|
|
|
| Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor
|
|
Author:
Date:
2017-01-20
[Abstract] Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is impaled by two microelectrodes, one for voltage sensing and the other one for current injection. Here we list a protocol that allows robust reconstitution of multi-component signaling pathways. This protocol has been used to study plant ion channels, including the SLAC1 channel (SLOW ANION CHANNEL-ASSOCIATED 1), in particular SLAC1 activation by either the protein kinase OST1 (OPEN STOMATA 1), Ca2+-dependent protein kinases (CPKs) or the ...
[摘要] 在非洲爪蟾卵母细胞中记录的双电极电压钳（TEVC）提供了一种强大的方法来研究离子通道蛋白的功能和调节。这种方法提供了一种众所周知的用于表征在非洲爪蟾卵母细胞中表达的离子通道或转运蛋白的工具。卵母细胞的质膜由两个微电极引起，一个用于电压感测，另一个用于电流注入。在这里，我们列出了一个允许多组分信号通路强制重组的协议。该方案已经用于研究植物离子通道，包括SLAC1通道（SLOW ANION CHANNEL-ASSOCIATED 1），特别是SLAC1通过蛋白激酶OST1（OPEN STOMATA 1），Ca 2 +依赖性蛋白激酶（CPK）或GHR1（GUARD细胞过氧化氢抗性1）跨膜受体样蛋白。显示了通过“单体”ABA（脱落酸）受体RCAR1 / PYL9（PYRABACT INRESISTANCE1 [PYR1] / PYR1-样[PYL] / ABA受体[RCAR]的调节因子]重建SLAC1阴离子通道的脱落酸活化的数据。通过共表达脱落酸信号核心的四个组分。该方案也适用于研究其他离子通道功能和调节机制，以及转运蛋白。背景 ...
|
|
|