Reconstitution of Chromatin by Stepwise Salt Dialysis
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Author:
Date:
2021-04-05
[Abstract] Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for a wide range of applications. Here, we describe simple and straightforward protocols for the reconstitution of chromatin by stepwise salt dialysis and the analysis of the chromatin by the micrococcal nuclease (MNase) digestion assay. Chromatin that is reconstituted with this method can be used for efficient homology-directed repair (HDR)-mediated gene edited with the CRISPR-Cas9 system as well as for biochemical studies of chromatin dynamics and ...
[摘要] [摘要]染色质而不是普通的DNA是介导细胞核中DNA定向过程的分子机器的天然底物。染色质可以重新构建d体外b ÿ使用不同的方法。盐渗析法产生的染色质由纯化的组蛋白和DNA组成。这种生物化学纯的染色质非常适合广泛的应用。在这里,我们描述了通过逐步盐透析和通过微球菌核酸酶(MNase)消化测定法对染色质进行分析的染色质重构的简单明了的协议。该复原用该方法染色质,可用于高效同源定向修复(HDR)介导的基因编辑编 使用CRISPR-Cas9系统,以及用于染色质动力学和功能的生化研究。
[背景]真核细胞中的DNA被组织成染色质。因此,理想情况下,DNA定向过程的分析(例如复制,重组,修复和转录)将使用染色质模板而不是纯DNA进行(Kadonaga,2019)。为此,染色质可通过使用ATP依赖性或ATP依赖性方法在体外从纯化的成分中重建(有关综述,请参见Lusser和Kadonaga,2004年)。
在这里,我们描述了染色质重建的特定方法,该方法在我们利用CRISPR-Cas9系统对细胞进行HDR介导的基因编辑研究中采用了(Cruz-Becerra和Kadonaga,2020年)。在这项工作中,我们发现相对于普通的(裸)DNA供体模板,通过使用染色质供体模板可以增强精确的HDR介导的DNA插入。我们还将这种方法用于染色质的生化分析,包括高迁移性N组(HMGN)蛋白(Rattner等,2009),染色质动力学(Torigoe等,2013),前核小体(Fei)的表征。等人,2015年),以及核小体失稳因子(NDF)(F ...
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Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
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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 ...
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Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression
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Author:
Date:
2017-06-05
[Abstract] A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo (Nicol et al., 2002; Chen et al., 2004). Emerging evidence indicates that plasmid bacterial backbone sequences are responsible for the transcriptional repression and this process is independent of CpG methylation (Chen et al., 2008). Dumbbell-shaped DNA vectors consisting solely of essential elements for transgene expression have been developed to circumvent these drawbacks. This novel non-viral vector has been shown ...
[摘要] 使用非病毒载体进行基因治疗的主要障碍是在postmitotic组织中转基因表达的持续时间短。以前的研究表明,即使载体DNA被保留,传代质粒的转基因表达也在递送后不久就下降到亚治疗水平,提示转录在体内沉默(Nicol等人)。 ,2002; Chen等人,2004)。新出现的证据表明质粒细菌骨架序列负责转录抑制,该过程与CpG甲基化无关(Chen等人,2008)。仅开发了用于转基因表达的必需元件的哑铃型DNA载体已被开发出来,以规避这些缺点。已经显示这种新的非病毒载体在体外和体内改善转基因表达(Schakowski等人,2001和2007)。在这里我们描述一种快速有效地生产最小化的表达小RNA的哑铃载体的新方法。简言之,将PCR扩增的启动子序列连接到化学合成的发夹RNA编码DNA模板以形成共价闭合的哑铃载体。这种新技术可以促进哑铃型载体用于临床前研究和人类基因治疗的应用。
背景 关于递送,小的载体大小有利地改善细胞外转运,包括通过细胞外基质网络的外渗和扩散以及细胞摄取和核扩散。已经开发了各种哑铃载体生产方法,包括产生表达小RNA的哑铃的方法,例如小发夹RNA(shRNA)和微小RNA(miRNA)(Schakowski等人,2001; ...
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