Bacterial Conjugation Protocol for Ruminant Mycoplasmas
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Author:
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2021-01-20
[Abstract] In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we describe an optimized conjugation protocol for the horizontal transfer of ICE or chromosomal DNA carrying antibiotic resistance markers (i.e., tetracycline, gentamicin, puromycin) from donor to recipient mycoplasma cells. Calculation of the ...
[摘要] [摘要]在无乳支原体中,已经描述了DNA转移的两个同时过程,它们需要直接的细胞间接触,并且类似于缀合。一种涉及整合共轭元件(ICE)的自我传递,而第二种涉及染色体DNA大小片段的水平转移。在这里,我们描述了一种优化的结合方案,用于从供体到受体支原体细胞水平转移带有抗生素抗性标记(即四环素,庆大霉素,嘌呤霉素)的ICE或染色体DNA 。详细介绍了共轭频率的计算,跨共轭物的选择和表征。该协议已与无乳分枝杆菌一起开发但已成功用于牛分枝杆菌,并可适应其他相关支原体物种。
[背景]共轭的,水平的DNA转移是微生物多样化的关键角色。通过促进细胞与细胞之间的紧密接触主动转移DNA (Lederberg和Tatum,1946),这种现象促进了从外部资源快速获取新性状。支原体(类柔膜)是无壁菌,其进化已经被认为是由基因损失仅减小驱动的基因组,并且其中水平基因转移(HGT )被长期被认为是边缘。在过去的十年中,比较基因组学分析重新审视了这种范例,并显示出(i)在共享同一宿主的支原体物种之间发生了HGTs事件(Sirand-Pugnet et al。,2007),以及(ii)整合和共轭的存在元件(ICE中)中的大量测序支原体基因组(Calcutt等人,2002; Marenda等人,2006; Dordet-弗里索尼等人,2013;拖期等人,2015; Meygret ...
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Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures
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Author:
Date:
2017-06-05
[Abstract] The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a ...
[摘要] 神经生物学领域中许多急性或慢性退行性疾病的体外模型的目的是评估神经元细胞的存活或损伤。细胞损伤与外细胞膜完整性的丧失和细胞质细胞蛋白的泄漏有关。因此,细胞培养上清液中细胞质酶的活性测定作为细胞损伤定量的实用工具(Koh和Choi,1987; Bruer等,1997)。乳酸脱氢酶(LDH)是一种无处不在表达的细胞溶质酶,由于蛋白质的半衰期很长,其稳定性很高(Hsieh和Blumenthal,1956; Koh和Cotman,1992; Koh等人)。 ,1995)。
背景 LDH在可逆的生物化学反应中催化丙酮酸和还原的烟酰胺偶氮二核苷酸(NADH)的乳酸盐和烟酰胺氨基茚三核苷酸(NAD +)的形成。 NADH在340nm的波长上具有吸收。这种动力学LDH活性测定的基础是由NADH降低引起的特定波长处的光密度降低。使用具有已知LDH活性的标准酶溶液计算上清液中LDH的量。不同的细胞密度或代谢活化率可能是混杂的;因此推荐LDH活性的正常化。这是通过评估外部细胞膜裂解后不抑制LDH活性的LDH活性(用0.5%Triton-X“完全杀死”)来实现的。最后,通过完全杀死的LDH活性的绝对LDH活性百分比表示细胞培养物中损伤或死细胞的发生率。
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A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
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Author:
Date:
2017-06-05
[Abstract] The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 ...
[摘要] 聚类规则间隔短回文重复(CRISPR)/ CRISPR相关蛋白9(Cas9)系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成:单导RNA(sgRNA)和Cas9。在特定靶序列引入双链断裂(DSB)需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的,因为它需要在体外转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA:CRISPR RNA(crRNA)和反式激活性crRNA(tracrRNA)进行靶向。这两种小RNA可以化学合成,这使得与sgRNA相比,这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而,已经开发了改进形式的Cas9以最小化离靶效应。例如,仅当两个引导RNA在规定的距离内结合相对的链时,切口酶型Cas9(nCas9)和FokI结构域融合的催化无活性的Cas9(FokI-dCas9; fCas9)才诱导DSB。在本协议中,我们描述了使用结合crRNA,tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂,而且可以降低脱靶效应的风险。
背景 ...
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