کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2089370 1545790 2006 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Enrichment of nucleofected primary human CD4+ T cells: A novel and efficient method for studying gene function and role in human primary T helper cell differentiation
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Enrichment of nucleofected primary human CD4+ T cells: A novel and efficient method for studying gene function and role in human primary T helper cell differentiation
چکیده انگلیسی

Identification of key factors mediating the differentiation of naïve CD4+ T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4+ T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2Kk cell surface marker followed by selection of H-2Kk positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2Kk positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4+ T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 310, Issues 1–2, 20 March 2006, Pages 30–39
نویسندگان
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