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Sterile syringe filter w/0.45 µm cellulose acetate membrane

0.22 μm滤器用于移液器辅助

Company: VWR
Catalog#: 28145-481
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Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
Author:
Date:
2020-06-20
[Abstract]  The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In addition, A. gossypii produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for A. gossypii that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 ... [摘要]  [摘要] CRISPR/Cas9系统是一种新的基因工具,可以精确地操作几乎所有的基因组序列。在这个协议中,我们使用一个特定的CRISPR/Cas9系统来操纵棉蚜。丝状真菌A.gossypii目前用于核黄素(vitamina B2)的工业生产。此外,棉蚜还产生其他高价值化合物,如叶酸、核苷和生物脂类。一个大型的分子工具箱可用于该真菌的基因组操作,包括基因靶向方法、异源表达模块的快速组装,以及最近一个适用于棉铃虫的单载体CRISPR/Cas9编辑系统,该系统允许实施无标记工程策略。CRISPR/Cas9系统包括RNA引导的DNA内切酶(Cas9)和与基因组靶区互补的引导RNA(gRNA)。Cas9核酸酶需要5′-NGG-3′三核苷酸,称为原间隔基序(PAM),在基因组靶区产生一个双链断裂(DSB),该断裂可以通过同源重组(HR)由合成的致突变供体DNA(dDNA)修复,从而引入一个特定的设计突变。适用于棉铃虫的CRISPR/Cas9系统极大地促进了这种工业真菌的基因组编辑。

[背景] ...

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells
Author:
Date:
2017-03-05
[Abstract]  Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type (Virdi et al., 2016). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that ... [摘要]  通过共焦激光扫描显微镜(CLSM)对植物中质体进行蛋白质靶向的研究可能由于许多来源的伪像而复杂化,其范围从体内蛋白质过度表达的误解,应激细胞中的假荧光,和细胞器错误识别。我们的研究集中在植物特异性基因MSH1上,其编码双重靶向蛋白,其在其表达中被调节并且位于特定质体类型的核内(Virdi等人,2016)。因此,我们的方法已被优化,以研究蛋白质双重靶向线粒体和质体,蛋白质表达的空间和时间调节和亚细胞定位,产生其他人可能对这些研究有用的方案和一组实验标准。

背景 植物中的蛋白质靶向行为受氨基末端前序列以及可影响亚组织定位行为的内部序列特征的影响(Baginsky和Gruissem,2004)。结合启动子驱动的表达空间和时间调节,蛋白质的活性可以通过时间和位置而非常精确和专一。在MSH1的情况下,这种核编码的植物特异性蛋白质是双重靶向线粒体和质体(Xu et al。,2011)。启动子特征将其表达指导到生殖,表皮和血管薄壁细胞(Virdi等人,2016)。内部蛋白质特征将其定位于线粒体和质体核,以及质体类囊体膜。使用这里描述的方法,通过激光扫描共聚焦显微镜大大促进了这些不寻常的蛋白质特征的发现。使用更传统的细分器分割方法,大部分细节将被忽略。

Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks
Author:
Date:
2016-05-20
[Abstract]  Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). ... [摘要]  呼吸道合胞病毒(RSV)属于包括许多临床相关病毒(例如人偏肺病毒和麻疹)的副粘病毒家族。 RSV感染可以在婴儿,老年人和一些免疫受损的成人中引起严重的疾病。在RSV复制期间,产生一系列截短形式的病毒基因组。这些截短的病毒基因组被称为缺陷型病毒基因组(DVG),并且由许多病毒产生(Lazzarini等人,1981; Rao和Huang,1982; Prince等人, ,1996; Sun等人,2015; Tapia等人,2013)。 DVG可以限制全长病毒的复制,并且是针对RSV的先天免疫应答的主要天然触发因子(Sun等人,2015; Tapia等人 ,2013)。在这里,我们详细讨论如何准备具有高或低含量的DVG的RSV股票,以及如何从富含缺陷病毒颗粒的RSV原液中纯化含有DVG的缺陷病毒颗粒。这些程序可用于制备实验室研究所需的病毒原液和有缺陷的病毒颗粒。简言之,在HEp2细胞中产生不同的RSV种群,其通常用于在实验室中扩增该病毒。为了产生具有高含量DVG的RSV原液,HEp2细胞用高多重感染(MOI)多次感染,然后使用梯度离心纯化含有DVG的病毒颗粒。这里描述的方法具有四个部分:1.具有低DVG含量(RSV-LD)的种子RSV原种的扩增,2.具有高DVG含量(RSV-HD)的原种的产生,3.通过梯度离心,4.纯化DVG的表征。

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