کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2093872 1401346 2016 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting
ترجمه فارسی عنوان
بیان ژن های بهبود یافته در سلول های بنیادی جنینی داکسی سیکلین القا های انتخاب شیمیایی مکرر و یا مرتب سازی سلول
کلمات کلیدی
azadC، 5-آزا 2'-دئوکسی سیتیدین؛ DOX، داکسی سیکلین. EGFP، افزایش پروتئین فلورسنت سبز؛ سلول های ES، سلول های بنیادی جنینی؛ ICE، القا سلول کاست exchangeInducible؛ ژن گزارشگر؛ mCherry به؛ سلول های بنیادی جنینی موش؛ سلول های تراریخته
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
چکیده انگلیسی


• DOX inducible ESC clones containing single copy transgenes show heterogeneous expression.
• The majority of the DOX non-responsive ESCs were transgene free.
• A small proportion of the ESCs fail to express the transgene due to epigenetic silencing.
• The non-inducible cells can be eliminated with chemical re-selection, or cell sorting.

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Stem Cell Research - Volume 17, Issue 2, September 2016, Pages 228–234
نویسندگان
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