Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells

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Abstract

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98 purity were obtained within 1week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells. © 2012 The Author(s).

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APA

Kacherovsky, N., Harkey, M. A., Anthony Blau, C., Giachelli, C. M., & Pun, S. H. (2012). Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells. Nucleic Acids Research, 40(11). https://doi.org/10.1093/nar/gks213

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