دورية أكاديمية
أعرض في EDS

CD-tagging-MS2: detecting allelic expression of endogenous mRNAs and their protein products in single cells.

التفاصيل البيبلوغرافية
العنوان: CD-tagging-MS2: detecting allelic expression of endogenous mRNAs and their protein products in single cells.
المؤلفون: Sheinberger J; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Hochberg H; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Lavi E; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Kanter I; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Avivi S; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Reinitz G; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Schwed A; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Aizler Y; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Varon E; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Kinor N; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel., Shav-Tal Y; The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel.
المصدر: Biology methods & protocols [Biol Methods Protoc] 2017 May 12; Vol. 2 (1), pp. bpx004. Date of Electronic Publication: 2017 May 12 (Print Publication: 2017).
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Oxford University Press Country of Publication: England NLM ID: 101693064 Publication Model: eCollection Cited Medium: Internet ISSN: 2396-8923 (Electronic) Linking ISSN: 23968923 NLM ISO Abbreviation: Biol Methods Protoc Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: [Oxford] : Oxford University Press, [2016]-
مستخلص: Discriminating between the mRNA and protein outputs of each of the alleles of an endogenous gene in intact cells, is a difficult task. To examine endogenous transcripts originating from a specific allele, we applied Central Dogma tagging (CD-tagging), which is based on a tag insertion into an endogenous gene by creation of a new exon. Previously, CD-tagging was used to tag endogenous proteins. Here we developed a CD-tagging-MS2 approach in which two tags were inserted in tandem; a fluorescent protein tag in conjunction with the mRNA MS2 tag used for tagging mRNAs in cells. A cell clone library of CD-tagged-MS2 genes was generated, and protein and mRNA distributions were examined and characterized in single cells. Taking advantage of having one allele tagged, we demonstrate how the transcriptional activity of all alleles, tagged and untagged, can be identified using single molecule RNA fluorescence in situ hybridization (smFISH). Allele-specific mRNA expression and localization were quantified under normal and stress conditions. The latter generate cytoplasmic stress granules (SGs) that can store mRNAs, and the distribution of the mRNAs within and outside of the SGs was measured. Altogether, CD-tagging-MS2 is a robust and inexpensive approach for direct simultaneous detection of an endogenous mRNA and its translated protein product in the same cell.
(© The Author 2017. Published by Oxford University Press.)
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فهرسة مساهمة: Keywords: allelic expression; gene expression; mRNA detection; stress granules; transcription
تواريخ الأحداث: Date Created: 20200313 Latest Revision: 20200928
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC6994078
DOI: 10.1093/biomethods/bpx004
PMID: 32161787
قاعدة البيانات: MEDLINE
الوصف
تدمد:2396-8923
DOI:10.1093/biomethods/bpx004