| DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
|
|
Author:
Date:
2017-06-05
[Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a web-based set of tools, named CRISPR RGEN tools (http://www.rgenome.net/), which provides all required tools from CRISPR target design to NGS data analysis.
[摘要] 我们通过使用无DNA的CRISPR成功地引入了目标克隆在赖氨酸衣藻中的目标基因敲除。在该协议中，整个工作流程的详细过程涵盖从初始目标选择CRISPR到使用下一代测序（NGS）技术的突变体分析。此外，我们介绍一种基于Web的工具集，名为CRISPR RGEN工具（ http：// www.rgenome.net/ ），其中提供了CRISPR目标设计到NGS数据分析的所有必需工具。
背景 我们最近报道（Baek等人，2016），使用预先组装的Cas9蛋白质指导RNA核糖核蛋白，模型绿色微藻（Chlamydomonas ...
|
|
|
| Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120
|
|
Author:
Date:
2016-08-05
[Abstract] Anabaena sp. strain PCC 7120 has long served as a model organism for investigating N2-fixation, photosynthesis, and various plant-type metabolic pathways and biofuel production, as well as cellular differentiation (Xu et al., 2008, Halfmann et al., 2014, Golden and Yoon, 2003). Since more than 30,000 sequenced bacterial genomes are currently available (Land et al., 2015), specific gene inactivation and analyses of the corresponding mutant’s phenotype have become powerful tools in elucidating the function of a target gene. Here we describe a protocol to inactivate a target gene in Anabaena sp. PCC 7120 using a single-crossover approach. This approach requires only one-step cloning of an internal fragment of a target gene into an ...
[摘要] 菌株PCC 7120长期充当用于研究N 2 - 固定，光合作用和各种植物类型代谢途径和生物燃料生产以及细胞分化的模式生物体（Xu等人，/em>。，2008，Halfmann等人，2014，Golden and Yoon，2003）。由于目前可获得超过30,000个测序的细菌基因组（Land等人，2015），特异性基因失活和相应突变体表型的分析已成为阐明靶基因功能的有力工具。在这里，我们描述了灭活anabaena sp中的靶基因的方案。 PCC 7120使用单交叉方法。该方法仅需要将靶基因的内部片段一步克隆到整合载体中以产生货物质粒。在货物质粒和鱼腥藻染色体之间的单次交换（同源重组）时，内源靶基因通过产生3'-和5'-缺失的片段而被破坏。该基因失活方案基于整合载体pZR606（Chen等人，2015），其可以广泛应用于其他蓝细菌物种以及其他原核生物中的基因失活。
|
|
|
| RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA
|
|
Author:
Date:
2015-05-20
[Abstract] Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).
[摘要] 免疫沉淀和随后的核酸分离允许研究蛋白质：核酸相互作用。 RNA结合蛋白免疫沉淀（RIP）用于分析蛋白质与mRNA的相互作用。 结合RIP与定量实时PCR（qRT-PCR）通过允许RNA结合蛋白与其靶mRNA的相互作用的定量评估以及这些相互作用在不同细胞设置中如何变化来进一步增强RIP技术。 在这里，我们描述RNA结合蛋白AUF1与几种不同的因素与衰老相关的分泌表型（SASP）（Alspach和斯图尔特，2013年），特别是IL6和IL8相关的免疫沉淀。 该协议最初发表在Alspach等人（2014）。
|
|
|