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Functional delivery of large genomic DNA to human cells with a peptide‐lipid vector
Author(s) -
White Robert E.,
WadeMartins Richard,
Hart Stephen L.,
Frampton Jon,
Huey Bryan,
DesaiMehta Ami,
Cerosaletti Karen M.,
Concan Patrick,
James Michael R.
Publication year - 2003
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.420
Subject(s) - transfection , plasmid , microbiology and biotechnology , biology , gene delivery , green fluorescent protein , genomic dna , genetic enhancement , dna , gene , genetics
Abstract Background Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin‐targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo , which may allow the transfer of genomic loci for gene therapy. Methods Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus ( NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence. Results We report a systematic study of the parameters influencing delivery of BAC‐based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin. Conclusions We show that LID vectors represent a promising tool for the transfer of complete genomic loci. Copyright © 2003 John Wiley & Sons, Ltd.