Nucleic Acids Research, Vol 25, Issue 10 1950-1956, Copyright © 1997 by Oxford University Press
A Baker and M Cotten
Molecular biology has many applications where the introduction of large
(>100 kb) DNA molecules is required. The current methods of large DNA
transfection are very inefficient. We reasoned that two limits to improving
transfection methods with these large DNA molecules were the difficulty of
preparing workable quantities of clean DNA and the lack of rapid assays to
determine transfection success. We have used bacterial artificial
chromosomes (BACs) based on the Escherichia coli F factor plasmid system,
which are simple to manipulate and purify in microgram quantities. Because
BAC plasmids are kept at one to two copies per cell, the problems of
rearrangement observed with YACs are eliminated. We have generated two
series of BAC vectors bearing marker genes for luciferase and green
fluorescent protein (GFP). Using these reagents, we have developed methods
of delivering BACs of up to 170 kb into mammalian cells with transfection
efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is
used as the carrier, thus eliminating the problems associated with viral
gene expression. The delivered DNA is linked to the carrier virus with a
condensing polycation. Further improvements in gene delivery were obtained
by replacing polylysine with low molecular weight polyethylenimine (PEI) as
the DNA condensing agent.
ARTICLES
Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier
Institute for Molecular Pathology (IMP), Dr BohrGasse 7, 1030 Vienna, Austria.
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