title: Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.
abstract: Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Matheson
authors.affiliation: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United Kingdom.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Peden
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, United Kingdom.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Lehner
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D060328:Magnetic Phenomena; D034741:RNA, Small Interfering / Q000235:genetics; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D034741:RNA, Small Interfering; C452700:streptavidin-binding peptide