Abstract
The transfer of large YAC DNA into human cells is a laborious procedure. High quality pulsed field gel purified DNA is required, which is easily sheared during manipulation before transfection or degraded in the endosome of the cell following transfection. NaCl and polyamines compact and prevent DNA from shearing, but may not consistently protect DNA after transfection. We investigated if other polycations such as poly-L-lysine (PLL) and polyethylenimine (PEI) could condense and protect large YAC DNA (up to 2.3 Mb) from being degraded after lipofection. DNA condensation was monitored by a gel retardation assay, and atomic force microscopy (AFM). DNA was retarded in the gel when complexed with high concentrations of PLL and PEI, indicating that DNA had condensed. However, AFM images of PLL–DNA complexes showed aggregates of DNA molecules resulting from incomplete condensation, whereas PEI–DNA complexes produced condensed particles approximately 30–60 nm. Exogenous PLL–DNA remained intact in 36% of positive clones after lipofection, whereas PEI–DNA was intact in 100% of positive clones. PEI is a better condensing reagent than PLL, protecting DNA from shearing and endosomal degradation, and assists in delivering YACs up to 2.3 Mb intact into human cells.
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Acknowledgements
We thank Dr Stephen Hart for an introduction to image collecting with the AFM, Dr Krish Narain (LOT-Oriel Ltd, Leatherhead, UK) for loan of an AFM and Dr Xiu Qiang Tong (Molecular Imaging) for help with MAC mode imaging. We are very grateful to Dr Robert Henderson and Dr Darren Ellis for access to an AFM and help with collecting the image in Figure 1a. We also thank Dr José Mea for helpful comments on the manuscript. The work was funded by SmithKline Beecham, the Medical Research Fund, University of Oxford and a Fellowship from the Wellcome Trust to ZL, and from the Deutsche Forschungsgemeinschaft to PM.
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Marschall, P., Malik, N. & Larin, Z. Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine. Gene Ther 6, 1634–1637 (1999). https://doi.org/10.1038/sj.gt.3300975
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DOI: https://doi.org/10.1038/sj.gt.3300975
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