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Verfasst von:Turra, Gino L. [VerfasserIn]   i
 Schneider, Luzia [VerfasserIn]   i
 Liedgens, Linda [VerfasserIn]   i
 Deponte, Marcel [VerfasserIn]   i
Titel:Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae
Verf.angabe:Gino L. Turra, Luzia Schneider, Linda Liedgens, Marcel Deponte
Jahr:2021
Umfang:11 S.
Teil:volume:241
 year:2021
 elocationid:111336
 pages:1-11
 extent:11
Fussnoten:Available online 6 November 2020 ; Gesehen am 10.09.2021
Titel Quelle:Enthalten in: Molecular and biochemical parasitology
Ort Quelle:Amsterdam : Elsevier, 1980
Jahr Quelle:2021
Band/Heft Quelle:241(2021), Artikel-ID 111336, Seite 1-11
ISSN Quelle:1872-9428
Abstract:Leishmania parasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited in Leishmania because of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Beneke et al. and the glmS ribozyme-based knock-down system in the model parasite Leishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in alternative media and identified supplemented MEM, IMDM and McCoy’s 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentration-dependent down-regulation of the episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.
DOI:doi:10.1016/j.molbiopara.2020.111336
URL:Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.

Volltext ; Verlag: https://doi.org/10.1016/j.molbiopara.2020.111336
 Volltext: https://www.sciencedirect.com/science/article/pii/S0166685120301006
 DOI: https://doi.org/10.1016/j.molbiopara.2020.111336
Datenträger:Online-Ressource
Sprache:eng
Sach-SW:cell culture
 CRISPR-Cas9
 knock-down
 ribozyme
K10plus-PPN:1753111471
Verknüpfungen:→ Zeitschrift

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