| Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
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Author:
Date:
2017-04-05
[Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a ...
[摘要] 可编程集群定期间隔短回归度（CRISPR）相关核酸酶9（Cas9）技术通过提供在所需位置切割基因组的有效方式，彻底改变了基因组编辑（Ledford，2015）。 在哺乳动物细胞中，DNA损伤触发易发生非同源末端连接（NHEJ）DNA修复机制。 然而，在DNA修复模板的存在下，可以发生同源性定向修复（HDR），导致病变部位的精确修复。 可以利用最后的方法，通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中，我们描述了使用重组腺相关病毒（rAAV）在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板（> 200个核苷酸）的递送。
尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告，敲门砖报告仍然落后。由于许多应用，产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在，并且在位点特异性双链（ds）DNA断裂被引入接近改变位点的基因组中后，HDR修复机制的激活。不同的模板可以传送到修复机器，范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链（ss）DNA寡核苷酸（Chen等人， ...
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| PBMC-MSC Co-cultures for Induction of Treg Generation
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Author:
Date:
2015-01-20
[Abstract] To assess the capacity of multipotent stromal cells (MSC) to induce the generation of Tregs, transwell co-cultures were performed as well as cultures with MSC-conditioned medium (CM). In short, peripheral blood mononuclear cells (PBMC) were co-cultured with allogeneic MSC or CM for one week followed by one week of culture in the absence of MSC.
[摘要] 为了评估多潜能基质细胞（MSC）诱导Treg产生的能力，进行Transwell共培养以及具有MSC条件培养基（CM）的培养。 简而言之，将外周血单核细胞（PBMC）与同种异体MSC或CM共培养一周，然后在不存在MSC的情况下培养一周。
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| siRNA Screening for Genes Involved in HSV-1 Replication
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Author:
Date:
2014-08-20
[Abstract] Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, ...
[摘要] 小干扰RNA（siRNA）是能够沉默转录后基因表达的小（通常为18-24个核苷酸）RNA分子，因此，它们提供了一种简单的方法，通过其可以容易地评估单个基因在复杂细胞系统中的作用。 由于siRNA易于实验使用，并且可以设计为靶向任何基因（包括病原体），它们的使用完全适合于并且容易地适应于高通量全基因组筛选方法学和一系列表型测定。 这里我们描述了使用大的siRNA文库（> 8,000个基因单独靶向）来筛选和鉴定功能上涉及人疱疹病毒（1型单纯疱疹病毒; HSV-1）（Griffiths）的复制的宿主因子， et al。，2013; Griffiths，2013）。
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