Oral Microbiome Characterization in Murine Models
|
Author:
Date:
2017-12-20
[Abstract] The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.
[摘要] 口腔微生物组被认为是口腔免疫应答的触发因素,特别是在炎性疾病牙周炎的形成中。 这里提出的协议旨在描述在稳定状态和扰动免疫或生理学的小鼠模型中的口腔微生物群。 在此,我们描述了鼠类口腔微生物群落取样程序,低生物量样品的处理和随后的基于16S rRNA基因测序的微生物群鉴定。
【背景】微生物组在调节组织特异性免疫应答(特别是在屏障部位)中起关键作用(Belkaid和Harrison,2017)。在这些屏障环境中,例如胃肠道和皮肤,选择的共生物显示能够驱动特定免疫细胞群体的发育(Ivanov等人,2009; Naik等人。,2012)。我们的工作最近开始探索口腔微生物组在剪裁组织免疫力方面的影响,尤其是在牙龈处,一个脆弱的口腔屏障部位(Abusleme和Moutsopoulos,2016; Dutzan等人,2017)。
在人类中,众所周知口腔中含有丰富多样的微生物(Human Microbiome Project,2012)。口腔微生物群落的改变与常见的口腔疾病,牙周炎(一种影响牙龈组织并导致组织损伤的炎症)有关(Griffen等人,2012; Abusleme等人。,2013; Moutsopoulos et al 。,2015)。迄今为止,动物模型已经有助于解决微生物组在各种生理和病理条件中的作用(Turnbaugh等人,2006; ...
|
|
Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
|
Author:
Date:
2017-05-20
[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for ...
[摘要] 为了实现克氏锥虫内源蛋白的C末端标记,我们使用Cas9 / pTREX-n载体(Lander等,2015)在3'末端插入特异性标签序列(3xHA或3xc-Myc)特定的感兴趣基因(GOI)。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增,并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记,加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M(Oberholzer等,2006)或pMOHX1Tag4H(Lander等,2016b)用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。 【背景】自从CRISPR / ...
|
|
Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells
|
Author:
Date:
2017-04-20
[Abstract] Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters.
This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following ...
[摘要] 细胞条形码可以通过单细胞谱系追踪来分离异种细胞群体中的克隆动力学。通过独特和可遗传的DNA条形码对造血干细胞和祖细胞(HSPC)进行标记,可以通过随后的移植和高通量测序,在单细胞水平上分化供体细胞异质性,分化潜力和谱系偏倚。此外,细胞条形码可以根据功能而不是免疫表型参数定义真正的造血干细胞(HSC)。  该协议描述了慢病毒细胞条形码的工作流程,追踪1450天后(dpc)胎肝(FL)Lineage-Sca+ cKit + (LSK)HSPC经过长期重建(图1)(Kristiansen等人,2016),但可以适应于选择的细胞类型或时间框架。
图1.实验工作流程摘要(Naik 等人,2013)
最初建立了细胞条形码技术来解决在体内移植造血细胞后的单细胞动力学,并且近年来在移植中对血细胞群体中功能异质性的认识有显着贡献(Schepers ,2008; Gerrits等人,2010; Lu等人,2011; Naik等人 ,2013; Verovskaya等人,2013; ...
|
|