Name: forward genetics screens using macrophages to identify toxoplasma gondii genes important for resistance to ifn-&#947;-dependent cell autonomous immunity
Uploaded: 2015-03-12T15:00:00
Description: Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃŽÂ³-Dependent Cell Autonomous Immunity at JoVE

Special thanks to Dr. Peter Bradley for the antibody to detect the T. gondii mitochondria. The work was supported by National Institute of Health Grants AI072028 and AI107431 to D.G.M and a generous donation to New York Medical College for the study of tropical medicine.

NOTE: All protocols that involve the use of animals were performed in accordance with the guidelines and regulations set forth by the New York Medical College&#8217;s Animal Care and Use Committee. 
NOTE: Detailed protocols for chemical mutagenesis 38, isolation of parasites by limiting dilution 38, isolation of murine bone marrow derived macrophages 39, growth of T. gondii in human foreskin fibroblast (HFF) cells and cyst production in macrophages and basic immunofluorescen...

The described protocol provides a non-biased approach that uses activation of murine bone marrow-derived macrophages and forward genetics to isolate T. gondii mutants with a defect in their ability to survive activation of infected macrophages. The phenotype of the mutants following macrophage activation typically falls into one of two broad categories: 1) The parasites appear intact but fail to replicate beyond 1 parasite per PV; 2) The parasites appear degraded and may be in spacious, amorphous PVs. The fact t...

Toxoplasma gondii (T. gondii) is an obligate intracellular, protozoal pathogen. It is the causative agent of toxoplasmosis, a health hazard in immunocompromised individuals. It is also the model system for other apicomplexan pathogens that infect humans including Cryptosporidium and Cyclospora. Toxoplasmosis is most commonly acquired through ingestion of food or water contaminated with the bradyzoite or oocyst stage of the parasite. Upon ingestion, these stages convert to the tachyzoite stage of the parasite that replicates within host cells and disseminates systemically. T cells, IFN-&#947; and, to a lesser extent, nitric oxide 1-4, are important for control of infection but are not capable of eliminating the disease, as a proportion of tachyzoites convert to the bradyzoite stage that are protected within tissue cysts resulting in a long-lived chronic infection. In fact, there are no therapeutics effective against the chronic cyst stage of the disease. Severe toxoplasmosis is most often due to the reactivation of persistent infection, with the bradyzoite stage of the parasite converting back to the rapidly replicating tachyzoite stage characteristic of primary and acute infection.
Early survival in the face of the innate immune response is important to allow the parasite to reach sufficient parasite numbers, as well as to reach distal sites, to enable establishment of chronic infection. T. gondii has evolved strategies to counteract host defense mechanisms that likely contribute to its ability to replicate and disseminate early in infection. First, T. gondii forms a unique PV during parasite invasion that is largely segregated from the endocytic and exocytic processes of the host cell compared to other intracellular pathogens 5-9. Also, like all successful intracellular pathogens T. gondii modifies its host cell to create a permissive environment for growth. This includes reprogramming host cell gene expression by altering host cell transcription factors including those important for regulating cell activation 10-15. ROP16 16-19, GRA15 20, GRA16 21 and GRA24 22 have all been shown to be important in regulating the transcriptional response and cell signaling cascades of host cells infected with T. gondii. Recent studies using genetic crosses between parasite strains with distinct phenotypes have been highly productive in identifying parasite genes that underlie parasite genotype-dependent traits including evasion of immunity related GTPases (IRGs) 16,19,23-26. In mice, immunity related GTPases (IRGs) are critical for the control of Type II and III genotypes of the parasite while the very virulent Type I genotypes have evolved mechanisms to evade the murine IRGs. However, it is also clear that the parasite has evolved mechanisms to evade antimicrobial mediators in addition to the IRGs and that some of these mechanisms may be conserved across parasite genotypes 27,28. In addition, very little is known about the critical mediators of cell autonomous immunity against T. gondii during human toxoplasmosis. Parasite genes important for resistance to mediators of cell autonomous immunity may also be important for survival during tachyzoite to bradyzoite conversion which can also be triggered by host immune responses. For example, nitric oxide at high levels can suppress parasite replication in infected macrophages but it can also stimulate tachyzoite to bradyzoite conversion resulting in cyst production30-32.
ToxoDB is a functional genomic database for T. gondii that functions as a critical resource for the field in terms of providing sequence information for the parasite genome and access to published and unpublished genomic scale data including community annotations, gene expression and proteomics data 33. Similar to many protozoal pathogens, the majority of the genome consists of hypothetical genes with no information available based on gene homology to provide insight into their potential functions. Thus, forward genetics is a powerful tool to identify novel parasite genes important for immune evasion, cyst conversion and other functions critical for parasite pathogenesis as well as for conversion between distinct developmental stages. An additional strength of forward genetics is that it can be used as a relatively non-biased approach to interrogate the parasite as to the genes that are important for specific tasks in pathogenesis, including immune evasion and cyst formation. Recent improvements in next generation sequencing for mutational profiling have made it a method of choice for identifying the responsible parasite genes from forward genetics studies using both chemical and insertional mutagenesis 34-37.
It is important to identify vulnerabilities in T. gondii that can be exploited to enhance the effectiveness of cell autonomous immune mechanisms against the parasite particularly those that may also be active against the resistant cyst stage. Toward this aim, an in vitro murine macrophage infection and activation model was developed to identify mutations in the parasite that specifically impair T. gondii fitness following activation of infected macrophages but not in na&#239;ve macrophages. This macrophage screen was used to interrogate a library of T. gondii insertional mutants in order to ultimately identify T. gondii genes important for resistance to nitric oxide 27,28. The isolation of a panel of T. gondii mutants with impaired resistance to activation of infected macrophages, particularly a marked sensitivity to nitric oxide, proved the utility of the screen to identify parasite genes important for resistance to mediators of cell autonomous immunity other than the resistance mechanisms described for the murine IRGs 28. Insertional mutagenesis has advantages over chemical mutagenesis in terms of generating a limited number of random mutations in each parasite clone and, in theory, easier identification of the site of mutation. However, identifying the genomic site of plasmid insertion in T. gondii insertional mutants, in practice, has been surprisingly difficult in many cases 37. Insertion of a plasmid into a gene is also likely to disrupt the function of a gene in contrast to chemical mutagenesis that typically results in single nucleotide changes. However, chemical mutagenesis with either N-ethyl-N-nitrosourea (ENU) or ethylmethane sulfonate (EMS) may offer an increased ability to analyze a larger portion of the parasite genome, compared to insertional mutagenesis, as it creates multiple single nucleotide polymorphisms (estimated at 10 -100) per mutant34,38. Moreover, recent advances in whole genome profiling has made it possible to use next generation sequencing to identify the most likely candidate genes responsible for the identified phenotype of a mutated parasite 34,38. Regardless of the mutagenesis approach, confirmation of the role of the parasite gene in resistance to macrophage activation ultimately requires gene deletion and complementation to fulfill molecular Koch&#8217;s postulates.
The ability to dissect the function of a gene by genetic manipulation of both the parasite and the macrophage is important as many of the genes identified via forward genetics in T. gondii, as well as other pathogens, are still characterized as hypothetical genes with little to no sequence homology to other proteins with known functions. The current paper outlines a general approach that can be used to identify whether the disrupted gene in a mutant is important for resistance to a known or unknown mediator of cell autonomous immunity. The initial analysis of host antimicrobial factors is performed by evaluating the survival of wild type and mutant parasites in macrophages from wild type mice versus those with specific gene deletions in inducible nitric oxide synthase (iNOS), gp-91 phox (NADPH oxidase), and specific immunity related GTPases (IRGs). This will determine if the identified parasite genes are important for resistance to nitric oxide, reactive oxygen intermediates or immunity related GTPases 28 respectively or if an unknown immune mechanism is involved. Activation of infected macrophages with both IFN-&#947; and LPS, described in the current protocol, results primarily in the isolation of parasite genes important for resistance to nitric oxide 28. The use of pharmacological agents that induce nitric oxide in the absence of macrophage activation (nitric oxide donors) confirmed that the majority of the genes identified were important for resistance to nitric oxide rather than nitric oxide in concert with additional mediators associated with macrophage activation 28.
Step one and two describe a forward genetics screen designed to isolate parasite mutants with a fitness defect following activation of infected bone marrow-derived macrophages in vitro. Step one describes a dose titration analysis to empirically determine a dose of IFN- &#947;&#160; and LPS to use for macrophage activation that reduces parasite replication but does not fully inhibit replication of the wild type T. gondii parental strain that is used for creation of the library of parasite mutants. Step two describes the forward genetic screen of the mutant clones in macrophages in 96-well plates. Step three outlines an approach to confirm the phenotype of each mutant identified in the screen of the 96 well plates and to evaluate whether the defect in each mutant affects parasite survival, replication, or cyst production in response to macrophage activation. Step four describes the use of bone marrow-derived macrophages from mice with deletions in specific antimicrobial pathways to identify the immune mediators to which the parasite mutant is specifically susceptible. Step five outlines an approach to determine if a parasite mutant is also compromised for in vivo pathogenesis as evaluated by cyst production in the brains of infected mice.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">Name of the material</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Hyclone</td>
			<td>SH3008101</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29101&#38;productId=3255471&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Thermo</td>
			<td>SH3091003</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=11737973&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DPBS</td>
			<td>Thermo</td>
			<td>SH3002802&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2434305&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType 
			=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone L-glutamine</td>
			<td>Thermo</td>
			<td>SH3003401&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3311957&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType 
			=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Pen strep</td>
			<td>Thermo</td>
			<td>SV30010&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&#38;productId=1309668 
			6&#38;catalogId=29104&#38;matchedCat 
			No=SV30010&#38;fromSearch=1&#38; 
			searchKey=SV30010&#38;highlightPro 
			ductsItemsFlag=Y&#38;endecaSearch 
			Query=%23store%3DRE_SC%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3DSV30010%2B%23searchType%3DPROD%23SWKeyList%3D%5B%5D&#38;xrefPartType=From&#38;savings= 0.0&#38;xrefEvent=1407777949003_0 
			&#38;searchType=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Hanks BSS</td>
			<td>Thermo</td>
			<td>SH3003002</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3064595&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>LIST biologicals</td>
			<td align="right">201</td>
			<td>http://www.listlabs.com/products-tech.php?cat_id=4&#38;product_id=81&#38;keywords 
			=LPS_from_%3Cem%3EEscherichia_coli%3C/em%3E_O111:B4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IFN-g</td>
			<td>Pepro Tech Inc</td>
			<td>50-813-664</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?itemdetail=&#39;item&#39;&#38;storeId=10652&#38; 
			productId=2988494&#38;catalogId=29 
			104&#38;matchedCatNo=50813664&#38; 
			fromSearch=1&#38;searchKey=murine+ifn+pepro+tech&#38;highlightProductsItemsFlag 
			=Y&#38;endecaSearchQuery=%23store%3DRE_SC%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3Dmurine%2Bifn%2Bpepro%2Btech%23searchType%3DPROD%23SWKeyList%3D%5B%5D&#38;xrefPartType=From&#38;savings 
			=0.0&#38;xrefEvent=1407778210608_ 
			12&#38;searchType=PROD&#38;hasPromo 
			=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber slides</td>
			<td>Thermo</td>
			<td align="right">177402</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2164545&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well optical plates</td>
			<td>Thermo</td>
			<td align="right">165306</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3010670&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">96-well tissue culture plates</td>
			<td></td>
			<td align="right">353072</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3158736&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Tissue culture flast T25</td>
			<td></td>
			<td align="right">156367</td>
			<td>https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&#38;productId=127039 
			67&#38;catalogId=29104&#38;matchedCat 
			No=12565351&#38;fromSearch=1&#38; 
			searchKey=156367&#38;highlightProdu 
			ctsItemsFlag=Y&#38;endecaSearchQu 
			ery=%23store%3DRE_SC%23nav%3D0%23rpp%3D25%23offSet%3D0%23keyWord%3D156367%23searchType%3DPROD%23SWKeyList%3D%5B%5D&#38;xrefPartType=From&#38;savings 
			=0.0&#38;xrefEvent=1407778974800_ 
			0&#38;searchType=PROD&#38;hasPromo 
			=0</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Ted Pella EM grade formaldehyde</td>
			<td></td>
			<td align="right">18505</td>
			<td>http://www.tedpella.com/chemical_html/chem3.htm#anchor267712</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Fisher</td>
			<td>BP151</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=3425922&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 - protein conjugation kit</td>
			<td>Life Technologies</td>
			<td>A20181</td>
			<td>http://www.lifetechnologies.com/order/catalog/product/A10235</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat serum</td>
			<td>MP Biomedicals</td>
			<td>ICN19135680</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2133236&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;crossRefData 
			=ICN19135680=2&#38;searchType 
			=PROD&#38;hasPromo=0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting media</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-conjugated dolichos</td>
			<td>Vector Labs</td>
			<td>FL-1031</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=188</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody to LAMP1</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
			<td>http://dshb.biology.uiowa.edu/LAMP-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysoTracker</td>
			<td>Life Technologies</td>
			<td>L-7526</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/L7526?ICID=search-product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL6 mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">664</td>
			<td>http://jaxmice.jax.org/strain/000664.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gp91 phox knock out mice</td>
			<td>Jackson Labaoratories</td>
			<td align="right">2365</td>
			<td>http://jaxmice.jax.org/strain/002365.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iNOS knock out mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">2609</td>
			<td>http://jaxmice.jax.org/strain/002609.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium nitroprusside</td>
			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2627727&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DETA NONOate</td>
			<td>ACROS Organics</td>
			<td>AC32865-0250</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2252389&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Monoclonal mouse anti-Toxoplasma gondii Ab</td>
			<td></td>
			<td>10T19A</td>
			<td>http://1degreebio.org/reagents/product/1069274/?qid=652947</td>
		</tr>
	</tbody>
</table>

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.

Toxoplasma gondii replicates freely in na&#239;ve macrophages and has a doubling time between 6-12 hr&#160;depending on the strain of the parasite. Figure 1 shows representative parasites in na&#239;ve versus activated bone marrow-derived macrophages. Figure 2 shows the general morphology of parasites in HFF host cells at 2, 4, 8, 16 and 32 parasites/PV. In the current protocol, the parasite is allowed to invade na&#239;ve macrophages and establish a nascent parasitophorous vacu...

Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃŽÂ³-Dependent Cell Autonomous Immunity at JoVE

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃƒÅ½Ã‚Â³-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.

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 ...

Education

Research

JoVE Core

JoVE Journal

Molecular Biology

Biology

Immunology and Infection

Evolution

Unit 2

Population Genetics

Analyzing Gene Expression and Function

SCIENTISTS IN ACTION

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

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 ...

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

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 ...

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

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 ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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

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

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

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

Genetic Manipulation in ÃƒÅ½Ã¢â‚¬Â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 ...

Forward Genetic Approaches in Chlamydia trachomatis

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...