PloS one, 2014-10, Vol.9 (10), p.e111437-e111437
2014
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Details
- Autor(en) / Beteiligte
- Titel
- Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification
- Ist Teil von
- PloS one, 2014-10, Vol.9 (10), p.e111437-e111437
- Ort / Verlag
- United States: Public Library of Science
- Erscheinungsjahr
- 2014
- Quelle
- Free E-Journal (出版社公開部分のみ）
- Beschreibungen/Notizen
- 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1932-6203eISSN: 1932-6203DOI: 10.1371/journal.pone.0111437Titel-ID: cdi_plos_journals_1619083794
- Format
- –
- Schlagworte
- Affinity, Affinity Labels - metabolism, Amino acids, Antibodies, Antigens, Beads, Biology and Life Sciences, Biotin, Cancer, Carrier Proteins - metabolism, CD4 antigen, CD4-Positive T-Lymphocytes - cytology, CD4-Positive T-Lymphocytes - metabolism, Cell culture, Cell lines, Cell Separation - methods, Cell surface, Cells (Biology), CRISPR, CRISPR-Cas Systems, Flow cytometry, Gene therapy, Genetic modification, Genetically modified organisms, Genome editing, Genomes, Genomics, HEK293 Cells, Hostages, Humans, Labeling, Lymphocytes, Lymphocytes T, Magnetic Phenomena, Mammalian cells, Medical research, Penicillin, Peptides, Proteins, Purification, Research and Analysis Methods, RNA, Small Interfering - genetics, Streptavidin, Surface markers, T cells, Time Factors, Transfection