Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine.
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.
Marschall
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P
Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford OX3 9DS, UK.
Malik
N
N
Larin
Z
Z
D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast; D018014:Gene Transfer Techniques; D006801:Humans; D018625:Microscopy, Atomic Force; D011094:Polyethyleneimine; D011107:Polylysine; D012965:Sodium Chloride
D011107:Polylysine; D012965:Sodium Chloride; D011094:Polyethyleneimine