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LipofectamineTM 2000 Transfection Reagent

Lipofectamine ® 2000转染试剂

Company: Thermo Fisher Scientific
Catalog#: 11668019
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Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
Author:
Date:
2017-08-05
[Abstract]  Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or ... [摘要]  唯一包含治疗相关序列的最小DNA载体对于新型治疗方案的发展具有很大的希望。哑铃型载体代表已经进入临床发展的晚期阶段的非病毒非整合DNA最小载体(Hardee等人,2017)。可接合的内含子和DNA核输入信号如SV40增强子序列是在质粒载体中发现多个应用以改善转基因表达的分子特征。然而,在哑铃中,由内含子引发的效应未被研究,基于DNA的核导入序列尚未发现应用,可能是因为哑铃载体在尺寸上不断被最小化。我们调查内含子和/或SV40增强子衍生序列对哑铃载体驱动的报告基因表达的影响。发现可拼接内含子的实现在所有研究的细胞系中无条件地增强基因表达。相反,SV40增强子的使用改善了细胞类型依赖性的基因表达。虽然这两个特征显着增加哑铃载体大小,但内含子和增强子或两者的组合都不会对基因表达产生负面影响。相反,与质粒或对照哑铃相比,这两个特征在一起改善了哑铃驱动的基因表达,高达160-或56倍。因此,强烈建议考虑用于哑铃矢量设计的内含子和SV40增强子。这种先进的设计可以促进哑铃型DNA载体的临床前和临床应用。
【背景】虽然许多基因已经使用哑铃形DNA载体表达,但大多数这些应用使用包括启动子,编码序列(CDS)和转录终止子的基本设计。一些载体包括嵌合内含子,然而,没有报道通过这种设计增强了转基因表达(Schirmbeck等人,2001)。在这里,我们研究了分子特征引发的效应,这些特征经常在哑铃驱动基因表达的质粒设计中得到应用:1.已知来自人β-珠蛋白基因剪接的嵌合内含子促进RNA加工,核出口,随后基因表达(Luo ...

Generation of a Cellular Reporter for Functional BRD4 Inhibition
Author:
Date:
2017-07-05
[Abstract]  The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many tumours. Even though BRD4 is more and more studied, its mechanism of action has not been fully described yet. Therefore, we aimed at generating a cellular reporter system to monitor BRD4 inhibition. Such reporter can be potentially used in high throughput chemical and genetic screenings, in order to uncover new possible BRD4 functional pathways. The deeper understanding of the mechanism of action of BRD4 activity will certainly help in developing ... [摘要]  普遍表达的含溴结构域的蛋白质4(BRD4)是表观遗传学读者,其将转录调节复合物募集到乙酰化染色质。 由于其在增强增殖中的作用,BRD4已经成为肿瘤学的治疗靶点,因为抑制这种蛋白质导致许多肿瘤的生长减少。 即使BRD4越来越多的研究,其行动机制尚未得到充分的描述。 因此,我们旨在产生细胞报告系统来监测BRD4抑制。 这种记录可以潜在地用于高通量化学和遗传筛选,以揭示新的可能的BRD4功能途径。 对BRD4活动作用机制的深入了解肯定有助于为所谓的BRD4依赖型癌症开发新的治疗策略。
【背景】表观遗传学领域的研究最近突出了BRD4在癌症进展中的中心作用。 BRD4是BET(溴结构域和终末外结构域)家族(Dey等人,2003; Filippakopoulos等人,2012; Wang)的乙酰赖氨酸阅读器, et al。,2012)能够结合启动子和增强子区域上的乙酰化组蛋白(Dey等人,2003; Filippakopoulos等人,2012; Nagarajan等人,,2014)。这种表观遗传学读者的作用机制在于通过招募几种转录因子,辅因子和RNA聚合酶II(RNApol II)来激活基因启动子和增强子,其导致调节,大部分增强某些靶基因的转录。已经描述了BRD4组蛋白模块发挥调控细胞周期进程的关键作用(Dey等人,2003; Wu和Chiang,2007; Yang等人, ...

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
Author:
Date:
2017-06-05
[Abstract]  Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an ... [摘要]  基于CRISPR的可编程RNA引导核酸酶(集群定期交织的短回文重复)-Cas(CRISPR相关蛋白)系统已被应用于各种类型的细胞作为强大的基因组编辑工具。通过使用激活诱导的胞苷脱氨酶(AID)代替CRISPR / Cas9系统的核酸酶活性,我们开发了一种用于靶向核苷酸替代(C至T或G至A)的基因组编辑工具,无供体DNA模板(图1 ; Nishida等人,2016)。这里我们描述Target-AID在哺乳动物细胞基因组中进行可编程点突变的详细方法。在这里描述了用于靶向中国仓鼠卵巢(CHO)细胞中的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶(HPRT)基因的具体方法作为实例,而该方法主要应适用于任何感兴趣的基因广泛的细胞类型。


图1. Target-AID及其可靶向位点的示意图。在指导RNA(gRNA)依赖性方式中,通过接头与nCas9(D10A)融合的PmCDA1在-21周围进行可编程胞苷突变至相对于哺乳动物细胞中非互补链上的PAM序列的-16位。可目标地点是根据以前的工作中观察到的有效的基础替代(> 20%)来确定的。
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