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T4 DNA Ligase

T4 DNA连接酶

Company: New England Biolabs
Catalog#: M0202
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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 ...

Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
Author:
Date:
2018-08-20
[Abstract]  Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis. A library of viral genomes, each containing a single randomly placed small insertion, is selected by passaging in cell culture and the insertion sites can be identified using Next Generation Sequencing (NGS). Here we describe a ... [摘要]  蛋白质标记是研究蛋白质功能的有效方法。然而,在病毒感染的情况下修饰正链RNA病毒蛋白可能特别困难,因为它们的紧密基因组和多功能蛋白意味着即使很小的变化也可以使病毒失活或减弱。尽管功能性标记病毒蛋白的靶向方法已经成功,但这些方法耗时且效率低。已经成功应用于几种RNA病毒的策略是全基因组转座子插入诱变。通过细胞培养中的传代选择病毒基因组文库,每个文库含有单个随机放置的小插入,并且可以使用下一代测序(NGS)鉴定插入位点。在这里,我们描述了用于登革病毒16681株血清型2的转座子诱变的方案。含有短随机放置插入物的突变登革病毒文库通过哺乳动物细胞传代,插入由有活力后代的NGS定位。该方案分为四个阶段:登革热cDNA克隆的转座子诱变,病毒基因组转染到允许细胞,分离病毒后代基因组和测序文库制备。

【背景】 ...

Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
Author:
Date:
2017-09-20
[Abstract]  Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We ... [摘要]  鉴于CRISPR(集群定期间隔短回归重复)编辑技术的出现,基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链(单向导)RNA(sgRNA)片段结合到相应的基因组靶序列,引发双链断裂。真核修复系统允许引入外源DNA,修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位(称为“多重”)。然而,CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代(“脱靶”)DNA位置。我们将安装的人造Cas9靶序列的使用(称为人造基因座上的Cas9复制)描述为允许(i)与单个sgRNA复用的酵母基因组中的用途; (ii)减少/消除可能的脱靶效应,以及(iii)精确控制预定目标序列的放置。
【背景】CRISPR(集群定期间隔回归重复)机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统(Jinek等,2012; Sorek等,2013)。这种生物技术需要使用来自化脓性链球菌(或othologous物种)的内切核酸酶(Cas9),单个RNA'引导'序列和外源供体DNA(如果需要)。仅在短短几年内,CRISPR / ...

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