title: Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.
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 1 week 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.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Nataly
authors.initials: N
authors.lastname: Kacherovsky
authors.forename: Michael A
authors.initials: MA
authors.lastname: Harkey
authors.forename: C Anthony
authors.initials: CA
authors.lastname: Blau
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Giachelli
authors.forename: Suzie H
authors.initials: SH
authors.lastname: Pun
mesh_terms: D000818:Animals; D060846:Cell Engineering / Q000379:methods*; D002460:Cell Line; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002470:Cell Survival; D004251:DNA Transposable Elements; D019281:Dimerization; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: C463061:AP20187; D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051496:Receptor, Fibroblast Growth Factor, Type 1; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus