Abstract
Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.
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Acknowledgments
We thank Dr. Neil Talbot at the USDA (Beltsville, MD) for providing the phEFnGFP plasmid and CT-1 cells and also for his assistance in establishing CT-1 cultures. We thank Dr. Ronald Zuckermann of the Molecular Foundry of the Lawrence Berkeley National Laboratory, Berkeley, CA, for generously supplying lipitoid reagent. This research was supported by Maryland Agricultural Experiment Station grants.
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Editor: T. Okamoto
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Schiffmacher, A.T., Keefer, C.L. Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells. In Vitro Cell.Dev.Biol.-Animal 48, 403–406 (2012). https://doi.org/10.1007/s11626-012-9525-9
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DOI: https://doi.org/10.1007/s11626-012-9525-9