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Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

التفاصيل البيبلوغرافية
العنوان: Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.
المؤلفون: Catherin Marin Mogollon, Fiona J A van Pul, Takashi Imai, Jai Ramesar, Séverine Chevalley-Maurel, Guido M de Roo, Sabrina A J Veld, Hans Kroeze, Blandine M D Franke-Fayard, Chris J Janse, Shahid M Khan
المصدر: PLoS ONE, Vol 11, Iss 12, p e0168362 (2016)
بيانات النشر: Public Library of Science (PLoS), 2016.
سنة النشر: 2016
المجموعة: LCC:Medicine
LCC:Science
مصطلحات موضوعية: Medicine, Science
الوصف: The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.
نوع الوثيقة: article
وصف الملف: electronic resource
اللغة: English
تدمد: 1932-6203
Relation: http://europepmc.org/articles/PMC5172577?pdf=render; https://doaj.org/toc/1932-6203
DOI: 10.1371/journal.pone.0168362
URL الوصول: https://doaj.org/article/d7b830b2b73247f48c74dcd02139f01a
رقم الأكسشن: edsdoj.7b830b2b73247f48c74dcd02139f01a
قاعدة البيانات: Directory of Open Access Journals
الوصف
تدمد:19326203
DOI:10.1371/journal.pone.0168362