| Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
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Author:
Date:
2017-08-05
[Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of ...
[摘要] 集群定期间隔短回归重复（CRISPR）和CRISPR相关（Cas）系统作为细菌和古菌中的适应性免疫系统，用于防御入侵病毒和质粒（Barrangou和Marraffini，2014）。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向（Jinek et al。，2012; Cong等人，2013; Mali ，2013; Zetsche等人，2015）。然而，真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9（SpCas9）靶向DNA接近的障碍，并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ （Knight等人，，2015; Hinz等人，2015; Horlbeck等人，2016年） ; ...
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| Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line
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Author:
Date:
2017-05-20
[Abstract] There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products.
[摘要] 有几个计算机程序尝试预测个体在剪接供体/受体位点的序列变异的功能影响，但实验确认是有问题的，没有携带变体的个体的RNA来源。借助于外显子捕获载体，例如pSPL3，研究人员可以通过在哺乳动物细胞中表达参考和变体小基因来测试剪接位点序列变化是否导致改变的RNA剪接，并分析所得RNA产物。
背景 我们希望通过实验测试在TEK基因中鉴定的两个剪接供体位点变体c.760 + 2T> C和c.3300 + 2delT的功能影响（Souma等人，2016）。通常情况下，携带这些序列变体的个体不能获得细胞或mRNA的样品，因此我们利用外显子捕获方法作为功能测试。来自患者的DNA样品可用于感兴趣的基因组区域的PCR扩增。如果患者gDNA样品不可用，也可以通过诸如基于PCR的定点诱变等方法将序列变体并入野生型序列。
＆NBSP;外显子捕获方法最初是为了鉴定长期基因组DNA中的未知外显子而开发的（Duyk等人，1990）。创建了pSPL3外显子捕获载体以提高外显子鉴定的效率和可靠性，并且还允许筛选更大的基因组片段（Church et al。，1994; Nisson等，1994）。 pSPL3载体含有由SV40启动子组成的小型人造基因，具有功能性剪接供体和受体位点的外显子 - 内含子 - ...
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| Infection Experiments (Hepatitis C Virus)
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Author:
Date:
2015-02-05
[Abstract] The establishment of a cell culture system for hepatitis C virus based on the JFH-1 strain and human hepatoma cell lines has been instrumental for the study of the viral replication cycle. The robustness of the JFH1-based cell culture models enabled many laboratories around the world to perform HCV infections in cell culture, accelerating the identification of cellular and viral targets to develop novel antiviral compounds. Although other robust infection systems based on different molecular clones and different cell lines have been developed since then, here we describe the protocols corresponding to infections with JFH-1 and JFH1-derived viruses carried out in our laboratory to produce virus stocks and persistently infected cell cultures. We also describe the experimental setups used to ...
[摘要] 基于JFH-1毒株和人肝癌细胞系的丙型肝炎病毒的细胞培养系统的建立已经有助于研究病毒复制周期。 基于JFH1的细胞培养模型的鲁棒性使世界上许多实验室在细胞培养物中执行HCV感染，加速细胞和病毒靶标的鉴定以开发新的抗病毒化合物。 虽然基于不同的分子克隆和不同的细胞系的其他强大的感染系统已经发展到今天，在这里我们描述了对应的感染JFH-1和JFH1衍生的病毒在我们的实验室生产病毒库存和持续感染细胞 文化。 我们还描述了用于确定病毒传播能力（多周期感染）以及解剖早期和晚期HCV感染（单周期感染）的实验设置。
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