我们要感谢她用流式细胞仪的协助下在加利福尼亚州国家灵长类动物研究中心的Abbie微调。这项研究是由国家过敏和传染病研究所（NIAID）美国国立卫生研究院（NIH）AI073745，AI080799，和AI078183提供资金支持。

<ol><li>启动协议之前，重要的是要确定，蚊子在实验中使用的细胞是健康的和〜80％汇合，如果贴壁（图1）。蔓生或不健康的细胞，会导致转染效率低，并会产生不一致的结果。 </li><li>前热身所有媒体在28℃水浴30分钟。 <ol class="lalpha"><li> A. stephensi MSQ43细胞需要E5介质：MEM，厄尔的W /谷氨酰胺，5％热灭活的FCS，0.2％D -葡萄糖，1％的青霉素，链霉素抗生素，1％非必需氨基酸。 </li><li> 冈比亚按蚊 SUA5B细胞需要S2介质：施耐德的果蝇培养基，5％热灭活的FCS，1％青霉素，链霉素抗生素</li></ol></li><li>解冻，并存储在冰块中的所有材料转染试剂，质粒和寡核苷酸。 </li><li>所有程序应在组织培养罩使用无菌技术进行。 </li><li>对于贴壁细胞（ 冈比亚按蚊 SUA5B A. stephensi MSQ43）吸过旧媒体不干扰细胞的情况下，添加一个新媒体的等量已预热至28 ° C，然后用清水冲洗干净，从底部的细胞容量瓶中，用吸管。 </li><li>细胞浓度应确定和调整到终浓度为1 × 10 6细胞/ mL。准备转染试剂材料的同时，细胞可在室温下保存。 </li><li>加入1毫升每6孔板以及蚊子细胞（1 × 10 6细胞/ mL）。设置一个盘子，每个实验条件下（见下文）。 表我是一个一般的转移液方案。表二列出了五个条件来测试使用特定的BFP - LNA组件的单一核苷酸的成功诱变。前两个，pGFP和pBFP，阳性和阴性对照，分别为。其余pBFPs实验条件下，与特定的BFP - LNA - ONS浓度增加。在步骤8到12，添加适当的试剂量为每一个条件。 </li><li>质粒DNA转移到无菌的1.5 ml离心管，加入适当体积的EC缓冲区。 <ol class="lalpha"><li> 1μL质粒DNA（1微克/μL），加99μL欧共体缓冲区。 </li><li> 1μLpGFP（1μg/μL），加99μL欧共体缓冲区。 </li><li> 1μLpBFP（1μg/μL），加99μL欧共体缓冲区。 </li><li> 1μLpBFP（1μg/μL）1μL，ON（μg/μL的），加98μL欧共体缓冲区。 </li><li> 1μLpBFP（1μg/μL）和5μL，ON（1μg/μL），加94μL欧共体缓冲区。 </li><li> 1μLpBFP（1μg/μL）和10μL，ON（1μg/μL），加89μL欧共体缓冲区。 </li></ol></li><li>然后慢慢加入强化剂使用DNA每1微克。 <ol class="lalpha"><li> 1μL质粒DNA，加入8μL的增强。 </li><li> 1微升pGFP，加入8μL的增强。 </li><li> 1μLpBFP，加8μL的增强。 </li><li> 1μL，pBFP和1μLON，添加16μL的增强。 </li><li> 1μL，pBFP和5μ​​LON，添加48μL的增强。 </li><li> 1μL，pBFP和10μL，ON，添加88μL的增强。 </li></ol></li><li>涡DNA /缓冲/增强的混合物为1秒，并确保所有的解决方案是在试管底部。然后在室温下孵育5分钟。 </li><li>新增每1μg质粒DNA和10秒的旋涡Effectene试剂。确认，所有的解决方案是在试管底部，然后在室温下孵育10分钟。 <ol class="lalpha"><li> 1μL质粒DNA，加入10μLEffectene（见表一）。 </li><li> 1微升pGFP，加入10μLEffectene（表二）。 </li><li> 1μLpBFP，加10μLEffectene（表二）。 </li><li> 1μL，pBFP和1μLON，加入20μLEffectene（表二）。 </li><li> 1μL，pBFP和5μ​​LON，加60μLEffectene（表二）。 </li><li> 1μL，pBFP和10μL，ON，添加110μL的Effectene（见表二）。 </li></ol></li><li>带给每管加入滴加DNA /缓冲/增强/ Effectene混合物加热的媒体，到1000μL的最终体积。 DNA可能沉淀在这一点上，如果媒体是增加太快。轻轻混合移液器向上和向下的3-4倍。 <ol class="lalpha"><li> 1μL质粒DNA，加882μL的媒体（见表一）。 </li><li> 1微升pGFP，添加882μL的媒体（见表二）。 </li><li> 1μLpBFP，加882μL的媒体（见表二）。 </li><li> 1μL，pBFP和1μLON，添加864μL的媒体（见表二）。 </li><li> 1μL，pBFP和5μ​​LON，添加792μL的媒体（见表二）。 </li><li> 1μL，pBFP和10μL，ON，添加702μL的媒体（见表二）。 </li></ol></li><li>下慢慢加入1毫升适当的DNA /增强/ Effectene混合物，每口井一次下降。 </li><li>板轻轻摇动，以确保细胞和转染混合物的均匀分布。 IncubatË细胞正常生长条件下。 </li><li>转染后的一天，轻轻地吸出媒体和慢慢取代等量的新鲜温暖的媒体。重要的是这样做时，不会干扰细胞层，在井底部。 </li><li>转染后两日内进行审查与pGFP阳性对照转染细胞，荧光显微镜下，以确定转染效率（图2）。 </li><li> SUA5B细胞生长非常迅速，将成为杂草丛生。四天后转薄这些细胞吸出旧媒体，并缓慢加入等量的新鲜温暖的媒体。 </li><li>添加新媒体的井后，从每口井的底部的所有细胞分离由pipeting上下，放弃一半的细胞混合物的体积（1毫升），并添加1毫升的新鲜温暖的媒体带来的最终体积为2毫升。 </li><li>日常检查细胞，以确定它们是健康的，而不是杂草丛生，必要薄。 </li><li>允许细胞生长正常情况下为五到七天后转染前收集分析。 </li><li>在五到七天后转，收集吸在所有井的旧媒体，每孔加入1毫升的无菌室温度PBS的细胞。 </li><li>从井底向上和向下吹打细胞分离，并转移至5 mL的管理单元第管的细胞混合，并立即通过流式细胞仪分析细胞。 </li></ol>代表性的成果<img src="/files/ftp_upload/2355/2355fig1.jpg" alt="图1" /> 未转染健康答： 图1。 stephensi MSQ43（A）和A.冈比亚 SUA5B（二）细胞在约80％汇合。 <img src="/files/ftp_upload/2355/2355fig2.jpg" alt="图2" /> 图2：A。 stephensi MSQ43（A）和A.冈比亚 SUA5B（二）细胞的转染与阳性对照的GFP表达质粒。 <img src="/files/ftp_upload/2355/2355fig3.jpg" alt="图3" /> 图3流式细胞仪检测数据，通过具体的BFP -锁定核酸修饰的寡核苷酸（LNA的ONS）以下的单核苷酸转染突变确认，BFP转换到绿色荧光蛋白。 SUA5B细胞转染与表达质粒绿色荧光蛋白（pGFP）作为阳性对照，表达质粒（pBFP）作为阴性对照的BFP，或与pBFP特定的BFP - LNA - ONS浓度的增加。 （一）从左至右流式细胞仪检测显示数据选通战略：一边向前与活细胞，碘化丙啶（PI）的阴性细胞，GFP阳性细胞5天以下转分散。 （二）图中（A）与低噪声放大器组件的pBFP和浓度的增加以下转染GFP阳性细胞的百分比。 <img src="/files/ftp_upload/2355/2355table1.jpg" alt="表1" /> 表一转染条件。 <img src="/files/ftp_upload/2355/2355table2.jpg" alt="表2" /> 表二，在图1中使用的转染条件。

<ol>
	<li>Corby-Harris, V. D. A., Watkins de Jong, L., Pakpour, N., Antonova, Y., Ziegler, R., Ramberg, F., Lewis, E. E., Brown, J. M., Luckhart, S. L., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+for+controlling+malaria+transmission+in+the+mosquito+Anopheles+stephensi.">A novel strategy for controlling malaria transmission in the mosquito Anopheles stephensi.</a> PLOS Pathogens. , (2010).</li><li>Braasch, D. A., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid+(LNA):+fine-tuning+the+recognition+of+DNA+and+RNA.">Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA.</a> Chem Biol. 8, 1-7 (2001).</li><li>Andrieu-Soler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transmission+of+targeted+gene+modification+using+single-stranded+oligonucleotides+with+flanking+LNAs.">Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.</a> Nucleic Acids Res. 33, 3733-3742 (2005).</li><li>Parekh-Olmedo, H., Drury, M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+nucleotide+exchange+in+Saccharomyces+cerevisiae+directed+by+short+oligonucleotides+containing+locked+nucleic+acids.">Targeted nucleotide exchange in Saccharomyces cerevisiae directed by short oligonucleotides containing locked nucleic acids.</a> Chem Biol. 9, 1073-1084 (2002).</li><li>Rhee, W. J., Santangelo, P. J., Jo, H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+accessibility+and+signal+specificity+in+live-cell+detection+of+BMP-4+mRNA+using+molecular+beacons.">Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons.</a> Nucleic Acids Res. 36, e30-e30 (2008).</li><li>Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+SNPs+with+molecular+beacons.">Genotyping SNPs with molecular beacons.</a> Methods Mol Biol. 212, 111-128 (2003).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat Biotechnol. 16, 49-53 (1998).</li><li>Blandin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+genetic+basis+of+resistance+to+malaria+parasites+in+Anopheles+gambiae.">Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae.</a> Science. 326, 147-150 (2009).</li><li>Niare, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+loci+affecting+resistance+to+human+malaria+parasites+in+a+West+African+mosquito+vector+population.">Genetic loci affecting resistance to human malaria parasites in a West African mosquito vector population.</a> Science. 298, 213-216 (2002).</li><li>Riehle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+malaria+infection+in+Anopheles+gambiae+is+regulated+by+a+single+genomic+control+region.">Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region.</a> Science. 312, 577-579 (2006).</li></ol>

在这里，我们目前在体外培养的方法和一个episomal基因目标有针对性地转换为一个单核苷酸的证据，A中的LNA组件的转stephensi和A 。 冈比亚细胞。 LNA - ON -介导的基因转化需要一个站点特定的DNA修复反应诱导;，我们的数据表明，蚊子细胞可以支持这个站点特定的突变机制。虽然这里介绍的方法是转换的episomal目标的基础上，我们的数据表明，低噪声放大器（LNA）的ON...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Effectene transfection reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>GFP control plasmid (pcDNA6.2-GW/EmGFP-miR-neg)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K4935-00</td>
<td>&nbsp;</td>
<tr>
<td>BFP plasmid (pcDNA5/FRT/TO)</td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LNA-ONs </td>
<td>&nbsp;</td>
<td>Gift from Dr. Concordet3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM, Earle's w/ glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11095</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum (FCS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Schneider&#x2019;s Drosophila medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11730</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140</td>
<td>&nbsp;</td>
<tr>
<td>10% D-glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5767</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin antibiotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides

疟原虫的寄生虫病，疟疾的病原体，导致超过250万，每年新感染者的感染按蚊蚊子叮咬传播。尽管有几十年的研究，还没有对疟疾疫苗，突出的新型控制策略的需要。一个创新的方法是使用基因改造的蚊子，有效控制疟疾寄生虫的传播。故意在蚊子的细胞信号通路的改变，通过有针对性的突变，已发现规范寄生虫的发展1。从这些研究中，我们可以开始转型，以确定潜在的靶基因。有针对性的突变历来依靠的一个靶基因和一个大的DNA分子之间的同源重组。然而，建设和使用等复杂的DNA分子稳定转化的细胞株的产生，是昂贵，费时，往往效率低下。因此，使用锁核酸修饰的寡核苷酸（LNA的组件）的战略提供了一个有用的替代引入episomal和染色体DNA基因的目标（2审查）人工单核苷酸替换。已使用的LNA - ON -介导的有针对性的突变引入点突变基因在培养细胞的酵母和小鼠3,4的兴趣。我们在这里展示，可用于低噪声放大器组件引入单一核苷酸开关按蚊和斯氏按蚊细胞在蓝色荧光蛋白（BFP）的表达绿色荧光蛋白（GFP）的表达变化，结果转episomal目标。这种转换表明，蚊子细胞中的靶基因的有效诱变可以介导的低噪声放大器组件和建议，这种技术可能适用，在体外和体内染色体目标诱变首次。

Watch this Scientific Journal Video about Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides at JoVE.com

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

寡核苷酸，可用于网站，专门替代的转靶基因的单核苷酸<em&gt;按蚊</em&gt;<em&gt;按蚊</em&gt;细胞。

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

transfection-mutagenesis-target-genes-mosquito-cells-locked-nucleic

Research

JoVE Journal

Immunology and Infection

10.3K 次观看.  University of California, Davis. 寡核苷酸，可用于网站，专门替代的转靶基因的单核苷酸按蚊按蚊细胞。

视频: Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.