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Novel membrane‐bound reporter molecule for sorting high producer cells by flow cytometry
Author(s) -
Helman Daniel,
ToisterAchituv Mira,
BarShimon Meirav,
Cohen Benny,
Otmi Issaschar,
Berger Nir,
Kalimi Doron,
Kimalov Boaz,
Medina Tali,
Sapir Ashi,
Rotemberg Ofer,
Zabavnik Natalia,
Zauberman Arie,
Smolarsky Moshe
Publication year - 2014
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.22308
Subject(s) - flow cytometry , sorting , membrane , cytometry , chemistry , microbiology and biotechnology , biophysics , biology , biochemistry , computer science , programming language
We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N ‐acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level. © 2013 International Society for Advancement of Cytometry