کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2830269 1163372 2008 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شناسی مولکولی
پیش نمایش صفحه اول مقاله
CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei
چکیده انگلیسی

The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Molecular and Biochemical Parasitology - Volume 157, Issue 1, January 2008, Pages 73–82
نویسندگان
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