Coupling Auxin-Inducible Degron System with Ultrastructure Expansion Microscopy to Accelerate the Discovery of Gene Function in Toxoplasma gondii

2021 ◽  
pp. 121-137
Author(s):  
Nicolas Dos Santos Pacheco ◽  
Dominique Soldati-Favre
Keyword(s):  
Toxoplasma Gondii ◽  
Gene Function

scholarly journals Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1390-1398 ◽  
Author(s):  
ELENA JIMÉNEZ-RUIZ ◽  
ELEANOR H. WONG ◽  
GURMAN S. PALL ◽  
MARKUS MEISSNER
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Gene Function ◽  
Function Analysis ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Advantages And Disadvantages ◽  
Gene Function Analysis ◽  
Tetracycline Inducible System

SUMMARYThe dissection of apicomplexan biology has been highly influenced by the genetic tools available for manipulation of parasite DNA. Here, we describe different techniques available for the generation of conditional mutants. Comparison of the advantages and disadvantages of the three most commonly used regulation systems: the tetracycline inducible system, the regulation of protein stability and site-specific recombination are discussed. Using some previously described examples we explore some of the pitfalls involved in gene-function analysis using these systems that can lead to wrong or over-interpretation of phenotypes. We will also mention different options to standardize the application of these techniques for the characterization of gene function in high-throughput.

scholarly journals Genetic Manipulation of the Toxoplasma gondii Genome by Fosmid Recombineering

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Sumiti Vinayak ◽  
Carrie F. Brooks ◽  
Anatoli Naumov ◽  
Elena S. Suvorova ◽  
Michael W. White ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Homologous Recombination ◽  
Gene Function ◽  
Genomic Dna ◽  
Gene Knockout ◽  
Single Copy ◽  
Fosmid Library ◽  
Chemical Mutagenesis ◽  
Parasite Development ◽  
Content Type

ABSTRACT Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite’s genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen. IMPORTANCE Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.

The importance of reverse genetics in determining gene function in apicomplexan parasites

Parasitology ◽  
1999 ◽  
Vol 118 (7) ◽  
pp. 53-61 ◽  
Author(s):  
M. SOETE ◽  
C. HETTMAN ◽  
D. SOLDATI
Keyword(s):  
Toxoplasma Gondii ◽  
Gene Function ◽  
Protein Trafficking ◽  
Cell Biology ◽  
Reverse Genetics ◽  
Regulation Of Gene Expression ◽  
Sequencing Project ◽  
Functional Roles ◽  
Obligate Intracellular ◽  
Intracellular Parasites

The phylum Apixomplexa includes obligate intracellular parasites that are of enormous medical and veterinary significance, as they are responsible for a wide variety of diseases including malaria, toxoplasmosis, coccidiosis, cryptosporidiosis, theileriosis and babesiosis. The EST sequencing projects in Toxoplasma gondii and the Plasmodium falciparum genome sequencing project have greatly accelerated gene discovery, revealing for example novel coding sequences restricted to the Apicomplexa. However, easy acquisition of sequence is almost useless if the function of any given gene cannot be tested. The establishment of transfection systems in Toxoplasma gondii, Neospora and in several Plasmodium species has provided us with the reverse genetics methods appropriate to the functional analysis of genes. Over the past few years, the discovery of novel genes coupled to the ability to introduce or modify genes has already contributed to a better understanding of cell biology and pathogenesis of these obligate intracellular parasites. Some insights into the complex processes of parasite invasion, differentiation, regulation of gene expression and protein trafficking are emerging although identification of the exact functional roles for many molecules is still awaiting more investigation. This review summarizes progress in this area. It also emphasises the tight link and synergy between Toxoplasma and malaria research. The use of reverse genetics does not guarantee the answer to gene function, so we can learn from both failed and successful experiments about how better and more efficiently to use ‘genomics’ to accelerate discoveries relevant to the understanding of parasitism by Apicomplexa.

Vorkommen und Genotypen von Toxoplasma gondii in der Muskulatur von Schaf, Rind und Schwein sowie im Katzenkot in der Schweiz

2012 ◽  
Vol 154 (6) ◽  
pp. 251-255 ◽  
Author(s):  
F. C. Frey ◽  
E. A. Berger-Schoch ◽  
C. D. Herrmann ◽  
G. Schares ◽  
N. Müller ◽  
...  
Keyword(s):  
Toxoplasma Gondii

Altered visual attention behavior of Toxoplasma gondii-infected individuals.

2019 ◽  
Vol 12 (4) ◽  
pp. 485-494
Author(s):  
Joaquim C. Rossini ◽  
Carolina S. Lopes ◽  
Fernanda P. Dirscherl ◽  
Deise A. O. Silva ◽  
José R. Mineo
Keyword(s):  
Toxoplasma Gondii ◽  
Visual Attention

Konnatale Toxoplasmose

2014 ◽  
Vol 14 (02) ◽  
pp. 101-106
Author(s):  
C. Feiterna-Sperling
Keyword(s):  
Toxoplasma Gondii

ZusammenfassungBei einer primären Toxoplasmose in der Schwangerschaft besteht für den Fetus das Risiko einer konnatalen Infektion durch diaplazentare Transmission von Toxoplasma gondii. Das Risiko einer fetalen Infektion nimmt dabei mit der Schwangerschaftsdauer zu, während die Schwere der Symptomatik mit zunehmendem Gestationsalter abnimmt. Bei den meisten infizierten Neugeborenen finden sich klinisch inapparente Infektionen, aber auch postnatal unauffällige Kinder sind einem Risiko von späteren Folgeschäden ausgesetzt. Neben neurologischen Entwicklungsstörungen ist vor allem das Risiko einer Retinochoroiditis von Bedeutung, die sich auch erst im späteren Leben manifestieren kann. Eine frühzeitige Erkennung einer Primärinfektion in der Schwangerschaft ist Voraussetzung, um durch eine frühzeitige anti-parasitäre Therapie, das Risiko einer fetalen Schädigung zu reduzieren. Durch eine post-natale Therapie kann vermutlich zusätzlich das Risiko der Langzeitkomplikationen gesenkt werden. Neugeborene mit Verdacht auf eine konnatale Toxoplasmose müssen sorgfältig hinsichtlich einer konnatalen Infektion untersucht werden und Säuglinge mit einer gesicherten Infektion benötigen langfristige Nachuntersuchungen hinsichtlich möglicher Spätschäden, insbesondere der Manifestation einer Retinochoroiditis.

The effect of cyclic AMP on the growth of Toxoplasma gondii in vitro

1990 ◽  
Vol 28 (2) ◽  
pp. 71 ◽  
Author(s):  
W Y Choi ◽  
H W Nam ◽  
J H Youn ◽  
D J Kim ◽  
W K Kim ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cyclic Amp

Analysis of antigenic domain of GST fused major surface protein (p30) fragments of Toxoplasma gondii

1996 ◽  
Vol 34 (2) ◽  
pp. 135 ◽  
Author(s):  
H W Nam ◽  
K S Im ◽  
E J Baek ◽  
W Y Choi ◽  
S Y Cho
Keyword(s):  
Toxoplasma Gondii ◽  
Surface Protein ◽  
Major Surface Protein ◽  
Major Surface ◽  
Antigenic Domain
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