עבודה זו מומנה על ידי איגוד הלב האמריקני המדען פיתוח גרנט 0635480N ואת המכונים הלאומיים לבריאות מענק מחקר AI081220. BIC נתמך על ידי קרן עין האבירים הטמפלרים מענק מחקר.

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אין לנו מה למסור.

<html> פרוטוקול תיאר מספק שיטה יעילה לבודד מוטנטים Toxoplasma עם מום יציאה. אנחנו מבודדים בהצלחה מוטציות לאורך השלבים השונים של מסלול יציאה, שחלקם יש פנוטיפ הפלישה כפול 13. השפעה על הפלישה ניתן לקבוע באמצעות מה שמכונה אדום ירוק assay, אשר מבדיל בין פלש ללא פלש טפילים י?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> ENU </td><td> Sigma-Aldrich </td><td> N3385 </td><td> 1 M לניירות ערך DMSO, החנות ב -20 ° C </td></tr><tr><td> EMS </td><td> Sigma-Aldrich </td><td> M0880 </td><td> 1 M לניירות ערך DMSO, החנות ב -20 ° C </td></tr><tr><td> Dextran סולפט </td><td> Sigma-Aldrich </td><td> D4911 </td><td></td></tr><tr><td> PDTC </td><td> Sigma-Aldrich </td><td> P8765 </td><td> 100 מ&quot;מ לניירות ערך PBS </td></tr><tr><td> הבדל מהירה </td><td> EMD כימיקלים </td><td> 65044-93 </td><td></td></tr><tr><td> מסנן בעל </td><td> קולמן בן הזוג </td><td> 540100 </td><td></td></tr><tr><td> 3 מיקרומטר פוליקרבונט המסנן </td><td> Whatmanשלייכר &amp; Schuell </td><td> 110612 </td><td></td></tr><tr><td> Hemocytometer </td><td> האוסר מדעי </td><td> 1475 </td><td></td></tr><tr><td> CO 2 חממות </td><td> יצרנים שונים </td><td></td><td> CO Humidified, 5% 2, ב 35, 37 ו - 40 ° C </td></tr><tr><td> מיקרוסקופ פלואורסצנטי </td><td> יצרנים שונים </td><td></td><td> הפוך באופן אידיאלי, תחום רחב במטרה שמן 63x או 100x </td></tr></tbody></table> HBSSc (לפי בלאק ואח&#39; 11.): <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 98.0 מ&quot;ל הנקס תמיסת מלח מאוזנת (מספר קטלוג Hyclone SH30588) </li><li style=";text-align:right;direction:rtl"> 100 μl 1M MgCl 2 (100 בסוף mM) </li><li style=";text-align:right;direction:rtl"> 100 μl 1M CaCl 2 (100 בסוף mM) </li><li style=";text-align:right;direction:rtl"> 2.0 מ&quot;ל 1M Hepes pH 7.3 (20 בסוף mM) </li><li style=";text-align:right;direction:rtl"> 84 מ&quot;ג NaHCO 3 (סוף 10 mM) </li></ul>

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

נפוצה, מחייבים תאיים, protozoan הטפיל Toxoplasma gondii גורם למחלות אופורטוניסטית ב חיסונית שנפרצו חולים וגורם למומים מולדים עם זיהום מולד. מחזור שכפול ממס מתאפיינת בשלושה שלבים: 1. הפלישה פעיל של תא פונדקאי גרעיני: 2. שכפול בתוך התא המארח, 3. יציאה פעילה מן התא המארח. מנגנון של יציאה היא יותר ויותר להיות מוערך כתהליך, ייחודי פיקוח הדוק, אשר עדיין ממעטים להבין ברמה המולקולרית. מסלולי איתות יציאה בבסיס אופיינו באמצעות סוכני פרמקולוגיים הפועלים על היבטים שונים של מסלולים 1-5. לפיכך, גורמים עצמאיים שונים של יציאה זוהו שכל להתכנס על שחרורו של Ca 2 + תאיים, סימן שהוא גם קריטי עבור הפלישה התא המארח 6-8. תובנה זו הודיע ​​הגן המועמד הגישה שהוביל identiאחת מהבעיות הנגזרות מהחלוקה של הצמח כמו סידן קינאז החלבון תלויה (CDPK) מעורב יציאה 9. בנוסף, פריצות הדרך האחרונות מספר יציאה הבנה נעשו באמצעות (כימי) גישות גנטיות 10-12. לשלב שפע של מידע תרופתי עם נגישות גנטי גדל והולך של Toxoplasma לנו לאחרונה הוקמה מסך המאפשר העשרה מוטציות טפיל עם פגם יציאה התא המארח 13. למרות mutagenesis כימי באמצעות N-ethyl-N-nitrosourea (ENU) או אתיל methanesulfonate (EMS) שימש במשך עשרות שנים בחקר הביולוגיה Toxoplasma 11,14,15, רק לאחרונה יש מיפוי גנטי של מוטציות שבבסיס פנוטיפים הופכים לשגרה 16 -18. יתר על כן, על ידי יצירת מוטציות רגישות טמפרטורה, תהליכים חיוניים ניתן גזור ואת הגנים הבסיסיים מזוהה באופן ישיר. מוטציות אלה מתנהגים כמו wild-type תחת טמפרטורה מתירנית (35 ° C), אבל לא מצליחים עמ &#39;roliferate בטמפרטורת כובל (40 ° C) כתוצאה של מוטציה הנדון. כאן אנו להמחיש שיטה חדשה ההקרנה פנוטיפי לבודד מוטנטים עם פנוטיפ הטמפרטורה רגיש יציאה 13. האתגר עבור מסכי היציאה היא להפריד egressed מן הלא egressed טפילים, אשר מורכבת על ידי מהיר מחדש הפלישה הדביקות הכללי של טפילים לתאי המארח. מסך יציאה הוקמה בעבר היה מבוסס על סדרה של צעדים מסורבלת biotinylation להפריד בין תאיים טפילים תאיים 11. שיטה זו גם לא ליצור מוטנטים מותנים וכתוצאה מכך פנוטיפים חלשים. השיטה המתוארת כאן מתגבר מצורף חזקה של egressing טפילים על ידי הכללת מתחרה glycan, סולפט dextran (DS), המונע טפילים יידבקו אל התא המארח 19. יתר על כן, טפילים תאיים נהרגים במיוחד את ידי pyrrolidine dithiocarbamate (PDTC), מה שמשאיר טפילים תאייםללא פגע 20. לכן, עם מסך פנוטיפי חדש במיוחד לבודד מוטנטים טפיל עם פגמים יציאה יזומה, כוחה של הגנטיקה כעת ניתן לפרוס באופן מלא לפענח את המנגנונים המולקולריים העומדים בבסיס התא המארח יציאה.

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

קדימה הגנטיקה היא שיטה רבת עוצמה לחשוף את הרמה המולקולרית של כמה<em&gt; Toxoplasma</em&gt; Egresses מהתא המארח שלה. הפרוטוקולים ניתנים כימית mutagenize טפילים, להעשיר את מוטנטים עם פגמים יציאה יזומה, ולאמת את הפנוטיפ של מוטציות משובטים.

מסך גנטית לבודד Toxoplasma gondii מארח תאים יציאה מוטציות

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

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Immunology and Infection

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

12.9K Views.  Boston College. קדימה הגנטיקה היא שיטה רבת עוצמה לחשוף את הרמה המולקולרית של כמה Toxoplasma Egresses מהתא המארח שלה. הפרוטוקולים ניתנים כימית mutagenize טפילים, להעשיר את מוטנטים עם פגמים יציאה יזומה, ולאמת את הפנוטיפ של מוטציות משובטים.

Video: 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

קבלת מטוהרים Toxoplasma gondii שביצים ידי Gradient כלוריד רציפה וצזיום

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

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.

Toxoplasma וול gondii ציסטה כינונה ב הופעל מח עצם הנגזרות המקרופאגים והגבלות Bradyzoite

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.

עכברים Transnuclear עם מוגדרים מראש תא קולטן T ספציפיות נגד Toxoplasma gondii לקבל ע&quot;י 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 ...

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.

באמצעות assay הפאן ויראלי microarray (Virochip) אל מסך דוגמאות קליניות עבור פתוגנים ויראליים

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.

הקמת תרבות הנוזלית מכוסית של Airway האנושי המקוטב-3 Calu תאי אפיתל ללמוד תגובת תא מארחת לפתוגנים נשימה<em&gt; במבחנה</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.

מניפולציה גנטית ב<em&gt; Δku80</em&gt; זנים לאנליזה גנומית תפקודית של<em&gt; Toxoplasma gondii</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.

שימוש בכימיה לחץ כדי למדוד את ההשפעה של זיהום ויראלי בסינתזת RNA Host-Cell

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.

קדימה גנטיקה מסכי שימוש המקרופאגים לזיהוי<em&gt; Toxoplasma gondii</emחסינות אוטונומית נייד להתנגדות לIFN-γ-Dependent&gt; גנים חשובים

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.

השימוש בטכניקת הכיסוי הרך-אגר למסך תרכובות מעכבות הופקו bacterially

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.

דרך לא פולשנית לבודד תאים פנוטיפ מן הקונגרס

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