この作品は、米国心臓協会の科学者は開発グラント0635480Nと健康研究助成金AI081220の国民の協会によって資金を供給された。 BICは、テンプル騎士団アイ財団研究助成金によってサポートされています。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+screen+to+dissect+Toxoplasma+gondii+egress.">Development of a screen to dissect Toxoplasma gondii egress.</a> Mol. Biochem. Parasitol. 171 (2), 97-103 (2010).</li><li>Pfefferkorn, E. R., Pfefferkorn, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii:+isolation+and+preliminary+characterization+of+temperature-sensitive+mutants.">Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants.</a> Exp. Parasitol. 39 (3), 365-376 (1976).</li><li>Pfefferkorn, E. R., Schwartzman, J. D., Kasper, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii:+use+of+mutants+to+study+the+host-parasite+relationship.">Toxoplasma gondii: use of mutants to study the host-parasite relationship.</a> Ciba. Found. Symp. 99, 74-91 (1983).</li><li>Striepen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+complementation+in+apicomplexan+parasites.">Genetic complementation in apicomplexan parasites.</a> Proc. Natl. Acad. Sci. U. S. A. 99 (9), 6304-6309 (2002).</li><li>White, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+rescue+of+a+Toxoplasma+gondii+conditional+cell+cycle+mutant.">Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant.</a> Mol. Microbiol. 55 (4), 1060-1071 (2005).</li><li>Gubbels, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+Genetic+Analysis+of+the+Apicomplexan+Cell+Division+Cycle+in+Toxoplasma+gondii.">Forward Genetic Analysis of the Apicomplexan Cell Division Cycle in Toxoplasma gondii.</a> PLoS Pathog. 4 (2), e36-e36 (2008).</li><li>Carruthers, V. B., Hakansson, S., Giddings, O. K., Sibley, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+uses+sulfated+proteoglycans+for+substrate+and+host+cell+attachment.">Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment.</a> Infect. Immun. 68 (7), 4005-4011 (2000).</li><li>Camps, M., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii:+selective+killing+of+extracellular+parasites+by+oxidation+using+pyrrolidine+dithiocarbamate.">Toxoplasma gondii: selective killing of extracellular parasites by oxidation using pyrrolidine dithiocarbamate.</a> Exp. Parasitol. 98 (4), 206-214 (2001).</li><li>Roos, D. S., Donald, R. G., Morrissette, N. S., Moulton, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+tools+for+genetic+dissection+of+the+protozoan+parasite+Toxoplasma+gondii.">Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii.</a> Methods Cell Biol. 45, 27-63 (1994).</li><li>Gubbels, M. J., Li, C., Striepen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+growth+assay+for+Toxoplasma+gondii+using+yellow+fluorescent+protein.">High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein.</a> Antimicrob. Agents Chemother. 47 (1), 309-316 (2003).</li><li>Carey, K. L., Westwood, N. J., Mitchison, T. J., Ward, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+approach+to+studying+invasive+mechanisms+of+Toxoplasma+gondii.">A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii.</a> Proc. Natl. Acad. Sci. U. S. A. 101 (19), 7433-7438 (2004).</li><li>Kafsack, B. F., Carruthers, V. B., Pineda, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modeling+of+Toxoplasma+gondii+invasion.">Kinetic modeling of Toxoplasma gondii invasion.</a> J. Theor. Biol. 249 (4), 817-825 (2007).</li><li>Hanash, S. M., Boehnke, M., Chu, E. H., Neel, J. V., Kuick, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+distribution+of+structural+mutants+in+ethylnitrosourea-treated+cultured+human+lymphoblastoid+cells.">Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.</a> Proc. Natl. Acad. Sci. U. S. A. 85 (1), 165-169 (1988).</li><li>Kafsack, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+membrane+disruption+by+a+perforin-like+protein+facilitates+parasite+exit+from+host+cells.">Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells.</a> Science. 323 (5913), 530-533 (2009).</li><li>Lovett, J. L., Marchesini, N., Moreno, S. N., Sibley, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+microneme+secretion+involves+intracellular+Ca(2+)+release+from+inositol+1,4,5-triphosphate+(IP(3))/ryanodine-sensitive+stores.">Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores.</a> J. Biol. Chem. 277 (29), 25870-25876 (2002).</li><li>Nagamune, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid+controls+calcium-dependent+egress+and+development+in+Toxoplasma+gondii.">Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii.</a> Nature. 451 (7175), 207-210 (2008).</li><li>Tomita, T., Yamada, T., Weiss, L. M., Orlofsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Externally+triggered+egress+is+the+major+fate+of+Toxoplasma+gondii+during+acute+infection.">Externally triggered egress is the major fate of Toxoplasma gondii during acute infection.</a> J. Immunol. 183 (10), 6667-6680 (2009).</li><li>Persson, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+receptor+ligation+or+exposure+to+perforin+trigger+rapid+egress+of+the+intracellular+parasite+Toxoplasma+gondii.">Death receptor ligation or exposure to perforin trigger rapid egress of the intracellular parasite Toxoplasma gondii.</a> J. Immunol. 179 (12), 8357-8365 (2007).</li></ol>

我々は、開示することは何もありません。

<html>記述されているプロトコルは、出口の欠陥とはトキソプラズマの変異体を単離するための効率的な方法を提供する。デュアル侵略の表現13を持っているそのうちのいくつかは我々が正常に、出力経路の様々なステップに沿って変異体を単離した。侵略の潜在的な影響は、差動抗体染色23,24によって非侵略寄生虫から侵入区別いわゆる赤緑のアッセイを用いて決定す?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> ENU </td><td> Sigma-Aldrich社</td><td> N3385 </td><td> -20℃でDMSO、ストア内の1 Mストック</td></tr><tr><td> EMS </td><td> Sigma-Aldrich社</td><td> M0880 </td><td> -20℃でDMSO、ストア内の1 Mストック</td></tr><tr><td>硫酸デキストラン</td><td> Sigma-Aldrich社</td><td> D4911 </td><td></td></tr><tr><td> PDTC </td><td> Sigma-Aldrich社</td><td> P8765 </td><td> PBS中100mMのストック</td></tr><tr><td> diffのクイック</td><td> EMDケミカルズ</td><td> 65044から93 </td><td></td></tr><tr><td>フィルターホルダー</td><td>コー​​ル·パーマー</td><td> 540100 </td><td></td></tr><tr><td> 3μmのポリカーボネートフィルター</td><td>ワットマンシュライヒャー＆シュル</td><td> 110612 </td><td></td></tr><tr><td>血球計</td><td> Hausser科学</td><td> 1475 </td><td></td></tr><tr><td> CO 2インキュベータ</td><td>さまざまなメーカー</td><td></td><td>加湿5％35 CO 2、37および40°C </td></tr><tr><td>蛍光顕微鏡</td><td>さまざまなメーカー</td><td></td><td> 63xまたは100倍の油を目的とした理想的には反転し、広視野</td></tr></tbody></table> HBSSc（ブラックらによると11。） <ul><li> 98.0ミリリットルハンクス平衡塩溶液（ハイクローンのカタログ番号SH30588） </li><li> 100μlの1MのMgCl 2（100mMの端） </li><li> 100μlの1MのCaCl 2（100mMの端） </li><li> 2.0ミリリットル1M Hepes緩衝液pH 7.3（20mMの終わり） </li><li> 84 mgを飽和NaHCO 3（10mMの端） </li></ul>

a genetic screen to isolate toxoplasma gondii host-cell egress mutants

広く、偏性細胞内、原虫寄生虫トキソプラズマ原虫は、免疫不全患者に日和見疾患を引き起こす先天性感染症に先天性欠損症を引き起こす。 1：溶菌複製サイクルは3つの段階によって特徴づけられる。核宿主細胞の積極的な侵攻2。宿主細胞内で複製、3。宿主細胞からのアクティブな出口。出口のメカニズムは、ますますまだ不十分分子レベルで理解されている、ユニークで高度に調節されたプロセスとして、高く評価されています。出口の根底にあるシグナル伝達経路は、経路1-5のさまざまな側面に作用する薬剤の使用によって特徴づけられている。このように、出口のいくつかの独立したトリガーはすべての細胞内Ca 2 +、また、宿主細胞の浸潤6-8重要である信号のリリースに収束するが同定されている。この洞察は、識別子につながった候補遺伝子アプローチを通知出口9に関与するカルシウム依存性プロテインキナーゼ（CDPK）のような植物のfication。さらに、理解の出口における最近のいくつかのブレークスルーが（化学）遺伝的アプローチを10-12を使用して行われています。 トキソプラズマの増加遺伝アクセシビリティと薬理学の豊富な情報を結合するために我々は最近、宿主細胞の出口13の欠陥を持つ寄生虫変異体の濃縮を可能にする画面を設立しました。 N-エチル-N-ニトロソウレア（ENU）またはエチルメタンスルホネート（EMS）を使用して、化学的突然変異誘発はトキソプラズマ生物学11,14,15の研究に何十年も使用されているが、ごく最近の基礎となる突然変異の遺伝子マッピングを持つ表現型は、ルーチン16になる-18。さらに、温度​​感受性変異体を生成することにより、本質的なプロセスは、解剖することができ、基礎となる遺伝子が直接識別されます。これらの変異体は、許容温度（35℃）下で野生型として動作するが、pに失敗する問題の突然変異の結果として、制限温度（40℃）でroliferate。ここでは、温度に敏感な出力表現型13に突然変異体を単離するための新たな表現型のスクリーニング方法を示しています。出力画面の課題は、宿主細胞に寄生虫の再侵攻、高速かつ一般的な粘りによって複雑にされた非egressed寄生虫からegressed分離することである。以前に確立された出力画面は、細胞外寄生体11から細胞を分離するビオチン化手順の面倒なシリーズに基づいていた。また、このメソッドは、弱い表現型の結果、条件付き変異体を生成しませんでした。ここで説明する方法は、宿主細胞19に付着する寄生虫を防ぐことが糖鎖のライバル、デキストラン硫酸（DS）を含めることによって寄生虫をegressingの強い愛着を克服しています。また、細胞外寄生体は、特に細胞内寄生体を残しピロリジンジチオカルバメート（PDTC）でオフに殺される20無傷。したがって、具体的に誘発される出口の欠陥で寄生虫の変異体を単離するための新たな表現型の画面で、遺伝学の力は完全に宿主細胞の出口の根底にある分子メカニズムを解明するために展開することができます。

Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

フォワード遺伝学はどのように分子レベルで解明するための強力な方法です<em&gt;トキソプラズマ</em&gt;その宿主細胞から出力する。プロトコルは化学的に寄生虫を突然変異を起こさせるために提供され、誘導される出口の欠陥を持つ変異体を濃縮し、クローニングした変異体の表現型を検証します。

分離するために遺伝学的スクリーニングトキソプラズマホストセル出口変異体

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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Research

JoVE Journal

Immunology and Infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

12.9K ビュー.  Boston College. フォワード遺伝学はどのように分子レベルで解明するための強力な方法ですトキソプラズマその宿主細胞から出力する。プロトコルは化学的に寄生虫を突然変異を起こさせるために提供され、誘導される出口の欠陥を持つ変異体を濃縮し、クローニングした変異体の表現型を検証します。

ビデオ: A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

高純度の取得トキソプラズマ</em不連続塩化セシウム勾配別>オーシスト

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

免疫・感染学

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

活性骨髄由来マクロファージとBradyzoite条件におけるトキソプラズマのシスト壁形成

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

ウイルス性病原体のために臨床サンプルをスクリーニングするためにパンウイルスマイクロアレイアッセイ（Virochip）を使用して、

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

呼吸器病原体に対する宿主細胞応答を研究するために偏ヒト気道上皮Calu-3細胞の液体に覆われた文化の確立<em&gt;インビトロ</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

の遺伝的操作<em&gt;Δku80</emの機能ゲノム解析のための&gt;株<em&gt;トキソプラズマ</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

フォワード遺伝学は、識別するためのマクロファージを使用した画面<em&gt;トキソプラズマ</emIFN-γ依存性細胞自律イミュニティへの抵抗のための重要な&gt;遺伝子

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

ソフト寒天オーバーレイ技術の使用は、細菌産生阻害する化合物をスクリーニングするために

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

結膜から細胞を単離して表現する非侵襲的方法

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...