A GRA2 minimal promoter improves the efficiency of TATi / Tet-Off conditional regulation of gene expression in Toxoplasma gondii

Sharifpour, Mohammad Farouq; Khadiv, Shadi; Meissner, Markus; McAllister, Milton M.

doi:10.1016/j.molbiopara.2021.111384

Cited by 3 publications

(3 citation statements)

References 19 publications

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“…There have been steady advances in genetic methods for Toxoplasma since the first transient and stable transfection of tachyzoites nearly 30 years ago [15,16] [20,21] and targeting specific chromosome breaks in or near target genes [22]. Because genetic deletion is unsuitable for required and essential genes, conditional knockdown methods have been developed that operate transcriptionally or post-translationally [23,24]. The high frequency of recombination achievable in laboratory strains like the Type I RH strain has permitted multiple forward genetic experiments with whole-genome CRISPR identification of required tachyzoite growth genes a recent example [25][26][27].…”

Section: Discussionmentioning

confidence: 99%

Engineering Toxoplasma transgenic tissue cysts

Hong

Brooks

Goerner

et al. 2022

Preprint

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Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central feature of reactivation. We have developed an innovative ex vivo model of bradyzoite recrudescence and applied this method to successfully modify Toxoplasma genes while avoiding the problems caused by continuous in vitro cell culture. We present four protocols required to engineer in vivo transgenic tissue cysts: 1) the reliable production of in vivo tissue cysts and excysted bradyzoites, 2) the use of fast-growing parasites from ex vivo bradyzoite infections to successfully generate transgenic tissue cysts in mice, 3) the cloning of transgenic bradyzoites via single cyst infections, and finally, 4) the long term cold storage and recovery of transgenic tissue cysts in brain tissue homogenates. We demonstrated these protocols by knocking out the Toxoplasma TgHXGPRT gene and the gene encoding the ApiAP2 transcription factor, AP2IX-9 in tissue cysts from mice. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite; host-dependent bradyzoite-to-bradyzoite replication.

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Section: Discussionmentioning

confidence: 99%

Engineering Toxoplasma transgenic tissue cysts

Hong

Brooks

Goerner

et al. 2022

Preprint

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“…Commonly used minimal promoters include S1O7 (SGA1 promoter with seven TetO sequences integrated) and S4O7 (SGA4 promoter with seven TetO sequences integrated) [46]. More recently, the use of the GRA2 minimal promoter has been reported to result in higher transcription of the target gene in the absence of tetracycline analogs such as anhydrotetracycline (ATc) and lower background expression when ATc is added, thus resulting in a 23-fold higher signal to noise ratio than that with the S1O7 promoter [47].…”

Section: Tunable Gene Transcription By Tetracycline Regulatable Promo...mentioning

confidence: 99%

Genetic Manipulation Toolkits in Apicomplexan Parasites

et al. 2022

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Apicomplexan parasites are a group of intracellular pathogens of great medical and veterinary importance, including Toxoplasma gondii and Plasmodium, which cause toxoplasmosis and malaria, respectively. Efficient and accurate manipulation of their genomes is essential to dissect their complex biology and to design new interventions. Over the past several decades, scientists have continually optimized the methods for genetic engineering in these organisms, and tremendous progress has been made. Here, we review the genetic manipulation tools currently used in several apicomplexan parasites, and discuss their advantages and limitations. The widely used CRISPR/Cas9 genome editing technique has been adapted in several apicomplexans and shown promising efficiency. In contrast, conditional gene regulation is available in only a limited number of organisms, mainly Plasmodium and Toxoplasma, thus posing a research bottleneck for other parasites. Conditional gene regulation can be achieved with tools that regulate gene expression at the DNA, RNA or protein level. However, a universal tool to address all needs of conditional gene manipulation remains lacking. Understanding the scope of application is key to selecting the proper method for gene manipulation.

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“…Minimal promoters used in Toxoplasma are derived from the gene promoters of TgSAG1 or TgSAG4 (Meissner et al., 2001 ). More recently, a GRA2‐derived minimal promoter was shown to allow more robust regulation (Sharifpour et al., 2021 ). The Tet‐off system has been first developed in Toxoplasma using the transactivator TATi‐1 (Trans‐Activator Trap identified), which was selected through a genetic screen based on random integration of TetR in T. gondii (Meissner et al., 2002 ) and was later shown to work as well in P. falciparum (Meissner et al., 2005 ).…”

Section: Regulated Gene Transcriptionmentioning

confidence: 99%

A toolbox for conditional control of gene expression in apicomplexan parasites

Briquet

Gissot

Silvie

2021

Molecular Microbiology

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The phylum Apicomplexa comprises more than 5,000 species, most of which are obligate intracellular parasites, including important pathogens for humans such as Plasmodium spp., Toxoplasma gondii and Cryptosporidium spp. Tremendous progress has been achieved over the past 20 years with the advent of genome sequencing and the availability of powerful bioinformatics interfaces to interrogate genome resources from many species and strains (Harb & Roos, 2015).An additional leap forward comes from the development of a variety of genetic tools to manipulate the genome of apicomplexan parasites. Reverse genetics has become a central approach to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies (Sexton et al., 2019). Genetic manipulation in apicomplexan parasites has been limited until now mainly to Toxoplasma and a few species of Plasmodium (including P. falciparum), but has been greatly facilitated over the past years through the diversification of selection procedures, including flow cytometry

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A GRA2 minimal promoter improves the efficiency of TATi / Tet-Off conditional regulation of gene expression in Toxoplasma gondii

Cited by 3 publications

References 19 publications

Engineering Toxoplasma transgenic tissue cysts

Engineering Toxoplasma transgenic tissue cysts

Genetic Manipulation Toolkits in Apicomplexan Parasites

A toolbox for conditional control of gene expression in apicomplexan parasites

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