PMID- 32161787 OWN - NLM STAT- PubMed-not-MEDLINE VI - 2 IP - 1 TI - CD-tagging-MS2: detecting allelic expression of endogenous mRNAs and their protein products in single cells. PG - bpx004 CI - © The Author 2017. Published by Oxford University Press. LA - eng PT - Journal Article PL - England TA - Biol Methods Protoc JT - Biology methods & protocols JID - 101693064 IS - 2396-8923 (Electronic) LID - 10.1093/biomethods/bpx004 [doi] FAU - Sheinberger, Jonathan AU - Sheinberger J AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Hochberg, Hodaya AU - Hochberg H AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Lavi, Erez AU - Lavi E AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Kanter, Itamar AU - Kanter I AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Avivi, Shira AU - Avivi S AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Reinitz, Gita AU - Reinitz G AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Schwed, Avital AU - Schwed A AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Aizler, Yuval AU - Aizler Y AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Varon, Eli AU - Varon E AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Kinor, Noa AU - Kinor N AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. FAU - Shav-Tal, Yaron AU - Shav-Tal Y AD - The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, 5290002, Israel. IS - 2396-8923 (Linking) OTO - NOTNLM OT - allelic expression OT - gene expression OT - mRNA detection OT - stress granules OT - transcription PMC - PMC6994078 LR - 20200928 DP - 2017 Jan DEP - 20170512 AB - 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.