| Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis
|
|
Author:
Date:
2018-02-05
[Abstract] Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the ...
[摘要] 莱茵衣藻（Chlamydomonas reinhardtii）是光合作用，代谢和鞭毛生物学的基础研究者。已经为这种藻类开发了多功能的工具箱（Fuhrmann等人，1999; Schroda等人，2000; Schroda，2006）。其中，正向遗传方法已经被广泛使用，主要是因为通过随机插入诱变产生了数十万个突变体的高效率，并且可以在第一代进行单倍体性质表型分析（Cagnon等人 2013，Tunçay et。 2013）。在正向遗传方法中应用高通量方法的主要瓶颈是鉴定与观察到的表型相关的遗传损伤。在该协议中，我们详细描述了最初在（González-Ballester等人，2005）中报道的限制性定点扩增PCR（RESDA-PCR）的改进版本。优化包括引物组合的优化，DNA聚合酶的选择，PCR循环参数的优化以及PCR产物直接测序的应用。这些修改使得获得特定的PCR产物变得更加容易，并且加速了亚克隆步骤以更快地获得测序数据。
【背景】除了限制酶位点定向扩增PCR（RESDA-PCR）（González-Ballester等人，2005）之外，还发现了其它几种分子技术。 包括Genome ...
|
|
|
| Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities
|
|
Author:
Date:
2015-11-20
[Abstract] The inflammatory response is essential to the reestablishment of cutaneous homeostasis following injury. In this context, leukocytes arrive at the wound site and orchestrate essential events in the wound healing process. Therefore, the quantification of specific subsets of inflammatory cells in the wound tissue is of considerable interest. The current protocol focus on a quantitative index of neutrophils and macrophages accumulation within skin lesions by measuring the specific activity of the marker enzymes Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG), respectively. MPO is present in high levels in the azurophilic granules of neutrophils and NAG in lysosomes of activated macrophages. These methods allow the indirect estimation of the abundance of neutrophils and ...
[摘要] 炎症反应对于损伤后皮肤稳态的再建立是必需的。 在这种情况下，白细胞到达伤口部位并协调伤口愈合过程中的重要事件。 因此，对伤口组织中炎性细胞的特定亚群的定量是相当感兴趣的。 通过测量标记酶髓过氧化物酶（MPO）和N-乙酰基-β-D-氨基葡萄糖苷酶（NAG）的比活性，目前的方案集中于皮肤损伤中嗜中性粒细胞和巨噬细胞积累的定量指数。 MPO以高水平存在于嗜中性粒细胞和NAG的嗜苯胺粒细胞中，在激活的巨噬细胞的溶酶体中。 这些方法允许间接估计积累到皮肤中的嗜中性粒细胞和巨噬细胞的丰度。
|
|
|
| Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
|
|
Author:
Date:
2014-09-05
[Abstract] RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 nuclease is directed by a short single guide RNA (gRNA or sgRNA) to generate double-strand breaks (DSB) at the specific sites of chromosomal DNA, thereby introducing mutations at the DSB by error-prone non-homologous end joining repairing. Cas9-gRNA recognizes targeted DNA based on complementarity between a gRNA spacer (~ 20 nt long leading sequence of gRNA) and its targeted DNA which precedes a protospacer-adjacent motif (PAM, Figure 1). In this ...
[摘要] 使用细菌II型簇定期间隔的短回文重复序列（CRISPR）相关核酸酶（Cas）的RNA指导的基因组编辑（RGE）已经作为用于在包括植物和作物物种的许多生物体中的基因组编辑的简单和通用工具而出现。在基于化脓性链球菌CRISPR-Cas9系统的RGE中，Cas9核酸酶由短的单引导RNA（gRNA或sgRNA）引导以在染色体的特定位点产生双链断裂（DSB） DNA，从而通过易错的非同源末端连接修复在DSB处引入突变。 Cas9-gRNA基于gRNA间隔区（约20nt的gRNA的前导序列）与其在原间质体相邻基序（PAM，图1）之前的靶DNA之间的互补性识别靶向DNA。在该协议中，我们描述了使用CRISPR-Cas9系统和农杆菌介导的转化的植物RGE的一般程序。该协议包括gRNA设计，Cas9-gRNA质粒构建和水稻RGE的突变检测（基因分型），可适用于其他植物物种。
|
|
|