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DPBS without calcium, magnesium

Dulbecco磷酸盐缓冲盐水

Company: GE Healthcare
Catalog#: SH30028.02
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High-throughput YO-PRO-1 Uptake Assay for P2X7 Receptors Expressed in HEK Cells
Author:
Date:
2018-07-20
[Abstract]  P2X7 receptors are extracellular ATP-gated ion channels that play broad physiological and pathological roles in animals (Sluyter, 2017). Activation of P2X7 receptors lead to the opening of membrane channels permeable for small cations like Na+ and Ca2+ as well as fluorescent dyes such as YO-PRO-1 (Alves et al., 2014). Taking advantage of this dye-permeability, YO-PRO-1 uptake assays have been widely used to probe P2X7 receptor activity (Surprenant et al., 1996; Rassendren et al., 1997; Karasawa et al., 2017). Here we describe a step by step protocol for a high-throughput YO-PRO-1 uptake assay using HEK293 cells expressing P2X7 receptors. This 3-day protocol is particularly suited for examining effects of small molecules and ... [摘要]  P2X7受体是细胞外ATP门控离子通道,在动物中发挥广泛的生理和病理作用(Sluyter,2017)。 P2X7受体的激活导致膜通道的开放可渗透小阳离子如Na + 和Ca 2 + 以及荧光染料如YO-PRO-1(Alves) et al。,2014)。 利用这种染料渗透性,YO-PRO-1摄取试验已被广泛用于探测P2X7受体活性(Surprenant et al。,1996; Rassendren et al。 ,1997; Karasawa et al。,2017)。 在这里,我们描述了使用表达P2X7受体的HEK293细胞进行高通量YO-PRO-1摄取测定的逐步方案。 这个为期3天的方案特别适用于检查小分子和突变对P2X7受体功能的影响。 该协议改编自我们之前发表的论文(Karasawa和Kawate,2016)。

【背景】P2X7受体打开可渗透阳离子的膜通道(Surprenant et al。,1996; Sluyter,2017)。虽然电生理学仍然是量化P2X7受体活性的金标准,但这种专门技术可能并不容易获得。此外,电生理学不适合高通量筛选,因为每次记录需要大量的时间和操作。因此,荧光分子的摄取是一种广泛使用的替代方法,特别是用于筛选多种条件/突变体(Cankurtaran-Sayar et al。,2009; Qu et al。 ...

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
Author:
Date:
2017-10-20
[Abstract]  In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter]

To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En ...
[摘要]  在该方案中,使用工程化的Cas9-核糖核蛋白(Cas9蛋白和sgRNA,一起称为Cas9-RNP)和金纳米颗粒制备用于将Cas9-RNP递送至细胞质和细胞核的纳米组件。 Cas9蛋白用N末端谷氨酸标签(E标签或En,其中n = E-标签中的谷氨酸数目,通常n = 15或20),C末端核定位信号(NLS) ,和C末端6xHis标签。 [Cas9En]

为了使用该方案,第一步是产生所需的材料(金纳米颗粒,重组Cas9En和sgRNA)。金纳米颗粒的实验室合成可能需要长达数周,但可以批量合成,可以使用多年,而不损害质量。可以从常规SpCas9基因(Addgene plasmid id = 47327)克隆Cas9En,并使用不属于该方案的标准实验室程序进行表达和纯化。类似地,可以使用来自模板基因(Addgene plasmid id = 51765)的体外转录实验室合成sgRNA,或者可以从各种来源购买。

一旦这些材料准备就绪,制作Cas9En-RNP复合物大约需要30分钟,并使得Cas9En-RNP /纳米纳米组件立即用于输送(图1)。完成交付(90-95%细胞质和核递送)在不到3小时内实现。后续编辑实验需要根据用户需要额外的时间。

报道了精氨酸功能化金纳米粒子(ArgNPs)(Yang等人,2011),重组Cas9En的表达和sgRNA的体外合成的合成(Mout ...

Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
Author:
Date:
2017-06-05
[Abstract]  A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to ... [摘要]  构建了一套Cas9和单引导CRISPR RNA表达载体。 需要一个非常简单的程序来制备具有高目标精度的特异性单向RNA表达载体。 由于在1细胞期的小鼠胚胎中已经检测到了合并的合子转录,所以将编码Cas9和GGTA1基因特异性单向RNA的质粒DNA载体微注射到合子原核中以确认 1细胞猪胚胎现象。 我们的研究结果表明,这些CRISPR / Cas9质粒引起的突变发生在猪胚胎的2细胞阶段之前和之后,表明除了体外转录的RNA的细胞质显微注射外,CRISPR / Cas9 DNA载体为产生基因敲除猪提供了有效的解决方案。

背景 由于最初发现了大肠杆菌基因组(Ishino)中的大肠杆菌基因组下游的32bp间隔的串联重复的高度保守的29个碱基对(bp)序列,等等,1987; Nakata等人,1989),在约50%至50bp的大小变化的短定期间隔重复序列的家族中发现约50%细菌和90%的古细菌(Makarova等人,2015)。根据它们的特征结构,Mojica(Mojica等人,2009)和Jansen(Jansen等人)引入了定期交织的短回文重复序列(CRISPR)的名称, ,2002),目前普遍使用。首先通过核苷酸序列比对(Jansen等人,2002)在CRISPR基因座的侧翼首先鉴定了一组CRISPR相关基因 cas1 ...

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