{{'Search' | translate}}
 

Falcon FACS tubes with 35 μMcell strainer

5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap

Company: BD
Catalog#: 352235
Bio-protocol()
Company-protocol()
Other protocol()

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
Author:
Date:
2017-04-05
[Abstract]  CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which ... [摘要]  基于CRISPR-Cas9的敲除策略越来越多地用于分析基因功能。然而,生物信号通路中的冗余和重叠功能可能需要产生多基因敲除细胞,这仍然是一个相对费力的过程。在这里,我们详细介绍了多色LentiCRISPR载体在人体细胞中同时产生单次和多次敲除的应用。我们提供了一个完整的方案,包括指导RNA设计,LentiCRISPR克隆,病毒生产和转导,以及排序和筛选敲除细胞的策略。该过程的有效性通过同时删除白血病细胞系中多达四个程序性细胞死亡介质和来自患者来源的急性淋巴细胞白血病异种移植物,其中单细胞克隆是不可行的。该协议允许任何具有基本细胞生物学设备的实验室,生物安全2级设备和荧光激活细胞分选功能,可在一个月内有效产生单基因和多基因敲除细胞系或原代细胞。

从对细菌基因组中被称为聚簇定期交织的短回文重复(CRISPR)的遗传元件的好奇的初步观察开始(Ishino等人,1987; Mojica等人,2000 )和随后在哺乳动物细胞中的基因编辑(Cong等人,2013; Mali等人,2013),CRISPR-Cas9已经成为廉价和有效的基因编辑。随着从烟草植物细胞到斑马鱼和原代人类细胞(Hsu等人,2014)的细胞系统的成功应用,CRISPR-Cas9可以通过短的20个核苷酸RNA序列的设计来引导在大基因组内的靶向DNA双链断裂(DSB)(Park等人,2016)。 ...

MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients
Author:
Date:
2017-03-20
[Abstract]  Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection.

In the following protocol, we describe a simple method that facilitates the identification of CD4+ ...
[摘要]  主要组织相容性复合物(MHC)四聚体已经使用二十年来检测,分离和表征各种病原体和肿瘤抗原特异性的T细胞。在人类免疫缺陷病毒(HIV)感染的背景下,抗原特异性CD8 +细胞已经在体外广泛研究,因为它们可以容易地被HIV肽 - 加载MHC I类四聚体。相比之下,HIV特异性CD4 + sup + T细胞的检测已被证明更具挑战性,因为CD4 + sup + T细胞的本质上较低的克隆扩增率以及优先艾滋病毒感染过程中艾滋病毒特异性CD4 + T细胞的消耗。 在以下协议中,我们描述了一种简单的方法,该方法有助于使用肽负载的MHC II类四聚体来鉴定HIV-1衣壳表位特异性的CD4 + / T细胞。可以分析四聚体标记的CD4 T细胞的细胞表面表型和/或FACS分选用于进一步的下游应用。成功检测特异性CD4 + / / T细胞离体的关键是选择导致高亲和力T细胞受体(TCR)的肽/ MHC II组合, (Benati等人,2016)。 MHC II四聚体阳性细胞的可靠检测的第二个关键点是系统地使用负载无关肽的对照四聚体,样品和对照管在相同的条件下进行处理。背景 在用抗PE微珠对四聚体-PE标记的细胞进行磁力富集后,在纯化的CD4 + T细胞中检测到罕见的HIV特异性MHC II四聚体阳性细胞(Seth等人, em>。,2005)。我们发现使用经验证的肽/ MHC ...

Knock-in Blunt Ligation Utilizing CRISPR/Cas9
Author:
Date:
2017-03-05
[Abstract]  The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example. [摘要]  将CRISPR / Cas9细菌免疫系统并入基因工程工具箱已经导致了几种用于基因组操作的新方法的开发(Auer等人,2014; Byrne等人,2015)。我们利用Cas9产生平端双链断裂的能力(Jinek等人,2012),以高度精确的方式通过开发非同源末端引物来引入外源DNA,加入DNA修复机械(Geisinger等人,2016)。该方案已成功应用于传统的永生化细胞系和人诱导多能干细胞。在这里,我们提出了使用HEK293细胞作为例子的敲入钝性连接的一般化方案。

背景 当我们概念化敲门钝性结扎(Geisinger等人,2016)时,开发用于CRISPR / Cas9的绝大多数方法都集中在提高同源重组的效率。然而,有一个例外:在斑马鱼中开发的同源性独立的基于质粒的敲入方法(Auer等人,2014)。这种方法,如敲入钝性连接,依赖于典型的非同源末端连接的机制,以线性化的,平端的双链DNA片段以高精度插入到基因组双链断裂中,核苷酸损失最小。这两种方法类似于为锌指核酸酶和被称为专性连接门控重组的TALEN开发的方法(ObLiGaRe; Maresca等人,2013),其依赖于产生相容的突出端以促进将靶DNA插入基因组。 ...

Comments