title: Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.
abstract: 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.
authors.affiliation: Interlab Ltd., an affiliate of Merck Serono S.A.
authors.forename: Daniel
authors.initials: D
authors.lastname: Helman
authors.forename: Mira
authors.initials: M
authors.lastname: Toister-Achituv
authors.forename: Meirav
authors.initials: M
authors.lastname: Bar-Shimon
authors.forename: Benny
authors.initials: B
authors.lastname: Cohen
authors.forename: Issaschar
authors.initials: I
authors.lastname: Otmi
authors.forename: Nir
authors.initials: N
authors.lastname: Berger
authors.forename: Doron
authors.initials: D
authors.lastname: Kalimi
authors.forename: Boaz
authors.initials: B
authors.lastname: Kimalov
authors.forename: Tali
authors.initials: T
authors.lastname: Medina
authors.forename: Ashi
authors.initials: A
authors.lastname: Sapir
authors.forename: Ofer
authors.initials: O
authors.lastname: Rotemberg
authors.forename: Natalia
authors.initials: N
authors.lastname: Zabavnik
authors.forename: Arie
authors.initials: A
authors.lastname: Zauberman
authors.forename: Moshe
authors.initials: M
authors.lastname: Smolarsky
mesh_terms: D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D003412:Cricetulus; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase
keywords: CHO; FACS; cell sorting; flow cytometry; high producer cells; reporter gene