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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Authors: Noelia Lander*
Noelia LanderAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
For correspondence: noelia@uga.edu
Bio-protocol author page: a4559
, Miguel A. Chiurillo*
Miguel A. ChiurilloAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4560
, Aníbal E. Vercesi
Aníbal E. VercesiAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4561
 and Roberto Docampo
Roberto DocampoAffiliation 1: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Affiliation 2: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
Bio-protocol author page: a4562
 (*contributed equally to this work),  date: 5/20/2017, 440 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2299.

[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned ...

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

Authors: Ashwin D’Souza
Ashwin D’SouzaAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3850
, Paulomi Sanghavi
Paulomi SanghaviAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3851
, Ashim Rai
Ashim RaiAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3852
, Divya Pathak
Divya PathakAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
Bio-protocol author page: a3853
 and Roop Mallik
Roop MallikAffiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
For correspondence: roop@tifr.res.in
Bio-protocol author page: a3854
,  date: 12/5/2016, 1308 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2056.

[Abstract] We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to ...

In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica

Authors: Merlyn Emmanuel
Merlyn EmmanuelAffiliation: Indian Institute of Science Education and Research Bhopal, Bhopal, India
Bio-protocol author page: a3003
 and Sunando Datta
Sunando DattaAffiliation: Indian Institute of Science Education and Research Bhopal, Bhopal, India
For correspondence: sunando@iiserb.ac.in
Bio-protocol author page: a3004
,  date: 3/20/2016, 1601 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1769.

[Abstract] Fluorescent matrix degradation assay is a popular and widely used assay in the field of invadopodium biology (Artym et al., 2009). Matrix remodeling and degradation can be observed under both physiological and pathological conditions. Cancer cells extensively remodel and degrade the underlying matrix ...

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

Authors: Jennifer Hirst
Jennifer HirstAffiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
For correspondence: jh228@cam.ac.uk
Bio-protocol author page: a2250
, Robert R Kay
Robert R KayAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
Bio-protocol author page: a2251
 and David Traynor
David TraynorAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
For correspondence: dt101@mrc-lmb.cam.ac.uk
Bio-protocol author page: a2252
,  date: 6/5/2015, 3314 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1485.

[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. ...

Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain

Authors: Valeria Bellini
Valeria BelliniAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2111
, Corinne Loeuillet
Corinne LoeuilletAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2112
, Céline Massera
Céline MasseraAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
Bio-protocol author page: a2113
, Marie-France Cesbron-Delauw
Marie-France Cesbron-DelauwAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
For correspondence: marie-france.cesbron@ujf-grenoble.fr
Bio-protocol author page: a2114
 and Pierre Cavaillès
Pierre CavaillèsAffiliation 1: Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, France
Affiliation 2: Université Joseph Fourier Grenoble 1, Grenoble, France
For correspondence: pierre.cavailles@ujf-grenoble.fr
Bio-protocol author page: a2115
,  date: 4/5/2015, 2638 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1439.

[Abstract] Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii, is characterized by a life-long chronic infection. The parasite is an efficient neurotropic infectious agent that establishes its “safe” life by forming intracellular cysts in chronically infected animals and humans. This protocol ...

A Gas Chromatography-Mass Spectrometry-Based Two Stage Assay for Measurement of in vitro myo-Inositol 3-phosphate Synthase (INO1) Activity

Authors: James I. MacRae
James I. MacRaeAffiliation 1: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia
Affiliation 2: The National Institute for Medical Research, The Ridgeway, London, UK
For correspondence: jmacrae@nimr.mrc.ac.uk
Bio-protocol author page: a2049
 and Malcolm J. McConville
Malcolm J. McConvilleAffiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia
Bio-protocol author page: a2050
,  date: 3/5/2015, 2815 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1418.

[Abstract] This method describes an in vitro assay for measuring INO1 enzyme activity (the conversion of glucose 6-phosphate to myo-inositol 3-phosphate) in cell-free extracts. The method was first described for Plasmodium falciparum cells in MacRae et al. (2014) and consists of two parts: Part 1 describes the ...

Preparation of Parasite Protein Extracts and Western Blot Analysis

Authors: Arlett Heiber
Arlett Heiber Affiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
,  date: 6/5/2014, 8241 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1136.

[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, ...

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

Authors: Arlett Heiber
Arlett HeiberAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
, Silke Retzlaff
Silke RetzlaffAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1409
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
,  date: 6/5/2014, 2944 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1137.

[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined ...

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress

Authors: Fiona Hackett
Fiona HackettAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1182
, Christine R Collins
Christine R CollinsAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1183
, Malcolm Strath
Malcolm StrathAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1184
 and Michael J Blackman
Michael J BlackmanAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
For correspondence: mblackm@nimr.mrc.ac.uk
Bio-protocol author page: a1185
,  date: 3/5/2014, 3353 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1055.

[Abstract] Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the ...

Protein Sample Preparation for Proteomic Analysis in Leishmania donovani

Authors: Alexandros Alexandratos
Alexandros AlexandratosAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
For correspondence: alexandratos@pasteur.gr
Bio-protocol author page: a1191
 and Despina Smirlis
Despina SmirlisAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
Bio-protocol author page: a1192
,  date: 3/5/2014, 3405 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1058.

[Abstract] Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional ...
1 2 

Preparation of Parasite Protein Extracts and Western Blot Analysis

Authors: Arlett Heiber
Arlett Heiber Affiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
,  date: 6/5/2014, 8241 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1136.

[Abstract] In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, ...

Plasmodium falciparum Rosette Formation Assay

Authors: Inès Vigan-Womas
Inès Vigan-WomasAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
For correspondence: ines.vigan-womas@pasteur.fr
Bio-protocol author page: a577
, Micheline Guillotte
Micheline GuillotteAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a576
 and Odile Mercereau-Puijalon
Odile Mercereau-PuijalonAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a308
,  date: 4/20/2013, 5726 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.412.

[Abstract] Rosetting, i.e. the capacity of red blood cells (iRBCs) infected with mature parasite stages to bind two or more uninfected red blood cells (RBCs) is a virulence factor of Plasmodium falciparum. This protocol describes an in vitro assay to monitor rosette formation by P. falciparum-infected red blood ...

Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages

Authors: Emma Iaconetti*
Emma IaconettiAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a157
, Brian Lynch*
Brian LynchAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a158
, Nathaniel Kim
Nathaniel KimAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
Bio-protocol author page: a159
 and Dana G. Mordue
Dana G. MordueAffiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA
For correspondence: dana_mordue@nymc.edu
Bio-protocol author page: a160
 (*contributed equally to this work),  date: 11/20/2012, 5369 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.289.

[Abstract] Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune ...

Glycophosphosphingolipid (GSPL) Purification Protocol

Author: Tripti De
Tripti DeAffiliation: Division of Infectious Disease and Immunology, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India
For correspondence: triptide@iicb.res.in
Bio-protocol author page: a175
,  date: 12/5/2012, 4493 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.299.

[Abstract] Glycosylated ceramide phosphorylinositol are present in many species of fungi and mushrooms and bacteria and parasitic organisms like leishmania. These are usually membrane raft associated and are not easily extracted by conventional methodologies. This extraction method gives higher yield of the glycolipid. ...

Plasmodium falciparum Rosette Disruption Assay

Authors: Micheline Guillotte
Micheline GuillotteAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
For correspondence: micheline.guillotte-blisnick@pasteur.fr
Bio-protocol author page: a576
, Odile Mercereau-Puijalon
Odile Mercereau-PuijalonAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a308
 and Inès Vigan-Womas
Inès Vigan-WomasAffiliation: Parasite Molecular Immunology Unit, Institut Pasteur, Paris, France
Bio-protocol author page: a577
,  date: 4/20/2013, 4189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.411.

[Abstract] Rosetting, i.e. the capacity of Plasmodium falciparum-infected red blood cells (iRBCs) to bind two or more uninfected red blood cells (RBCs) is associated with severe malaria in African children. Disruption of rosettes using small soluble inhibitors or specific antibodies is viewed as an interesting ...

EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)

Author: Jianyang Wang
Jianyang WangAffiliation: Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA
For correspondence: jwang117@jhu.edu
Bio-protocol author page: a636
,  date: 6/20/2013, 3808 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.799.

[Abstract] Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations ...

Protein Sample Preparation for Proteomic Analysis in Leishmania donovani

Authors: Alexandros Alexandratos
Alexandros AlexandratosAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
For correspondence: alexandratos@pasteur.gr
Bio-protocol author page: a1191
 and Despina Smirlis
Despina SmirlisAffiliation: Molecular Parasitology Department, Hellenic Pasteur Institute, Athens, Greece
Bio-protocol author page: a1192
,  date: 3/5/2014, 3405 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1058.

[Abstract] Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional ...

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress

Authors: Fiona Hackett
Fiona HackettAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1182
, Christine R Collins
Christine R CollinsAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1183
, Malcolm Strath
Malcolm StrathAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
Bio-protocol author page: a1184
 and Michael J Blackman
Michael J BlackmanAffiliation: Division of Parasitology, MRC National Institute for Medical Research, London, UK
For correspondence: mblackm@nimr.mrc.ac.uk
Bio-protocol author page: a1185
,  date: 3/5/2014, 3353 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1055.

[Abstract] Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the ...

Dictyostelium Cultivation, Transfection, Microscopy and Fractionation

Authors: Jennifer Hirst
Jennifer HirstAffiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK
For correspondence: jh228@cam.ac.uk
Bio-protocol author page: a2250
, Robert R Kay
Robert R KayAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
Bio-protocol author page: a2251
 and David Traynor
David TraynorAffiliation: MRC Laboratory of Molecular Biology, Cambridge, UK
For correspondence: dt101@mrc-lmb.cam.ac.uk
Bio-protocol author page: a2252
,  date: 6/5/2015, 3314 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1485.

[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. ...

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

Authors: Arlett Heiber
Arlett HeiberAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1407
, Silke Retzlaff
Silke RetzlaffAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Bio-protocol author page: a1409
 and Tobias Spielmann
Tobias SpielmannAffiliation: Parasitology Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
For correspondence: spielmann@bni-hamburg.de
Bio-protocol author page: a1408
,  date: 6/5/2014, 2944 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1137.

[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined ...
1 2 
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Hot protocols of the week
Preparation of Parasite Protein Extracts and Western Blot Analysis
Plasmodium falciparum Rosette Formation Assay
Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays
Protein Sample Preparation for Proteomic Analysis in Leishmania donovani
Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress
Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
Plasmodium falciparum Rosette Disruption Assay
In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica

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