کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2089596 1545788 2006 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Floxed reporter genes: Flow-cytometric selection of clonable cells expressing high levels of a target gene after tamoxifen-regulated Cre–loxP recombination
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Floxed reporter genes: Flow-cytometric selection of clonable cells expressing high levels of a target gene after tamoxifen-regulated Cre–loxP recombination
چکیده انگلیسی

Tamoxifen treatment allows MerCreMer fusion recombinase to localize to the nucleus where MerCreMer can excise a floxed inhibitory DNA segment, thereby activating the expression of a downstream gene. This excision is irreversible, and it is therefore difficult to predict which non-activated clones will express the gene at high levels after recombination. We transfected a vector using HLA-A2.1 as floxed inhibitory DNA element and its expression level as surrogate marker predicting future expression of the attenuated downstream target gene. The target gene encoded an EGFP-linked fusion protein. In the unsorted population, 6% of the cells expressed the transfected target gene after recombination and less than 10-fold higher than the population before recombination. However after flow-cytometric selection for high HLA-A2.1 expression, 47% of the cells expressed the target gene after recombination and at levels 37-fold higher than the sorted population before recombination. 58% of the clones were capable of expressing the fusion protein and some over 200-fold above background of untransfected cells and greater than 20-fold higher levels of expression than before recombination. We describe an efficient method to select for clones expressing high levels of a target gene after tamoxifen regulated Cre–loxP recombination. Other floxed reporter genes should be equally useful.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 312, Issues 1–2, 30 May 2006, Pages 201–208
نویسندگان
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