ArticleMethods in molecular biology (Clifton, N.J.)2023
Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.
Article in Methods in molecular biology (Clifton, N.J.), 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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3 citing papers in PubMed, 2 citations in OpenAlex.
- Integrating HTS and CRISPR/Cas for next-generation nucleic and non-nucleic acid diagnostics.Molecular genetics and genomics : MGG · 2026Review
- Advances in Serum-Free Suspension Culture Technology for Animal Cells and Their Applications.Vaccines · 2025Review
- Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology.Antibodies (Basel, Switzerland) · 2023Article
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6 authors at 3 institutions in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.
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