Formation and use of water-soluble lipopolymer-pDNA and lipopeptide-pDNA complexes.
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| Abstract |
Water-soluble lipopolymers and lipopeptides are nonviral gene-transfer reagents that combine the advantages of lipids, which increase permeability of DNA through cell membranes, with the DNA-condensing and enhanced endosomal release properties of polycations. This protocol describes the synthesis and characterization of water-soluble lipopolymer/lipopeptide-DNA complexes and their use in transfection of cultured cells and injection into tumor-bearing mice. Lipopolymers are formed by conjugating cholesteryl chloroformate with the primary or secondary amines of 1800-Da branched polyethylenimine (PEI); the use of the 1800-Da PEI avoids the cytotoxicity problems associated with higher molecular-mass PEI. Lipopeptides are composed of a human protamine-derived peptide that has been incubated with a reaction of O-(N-succinimidyl)-N,N,N',N',-tetramethyluronium tetrafluoroborate with lithocholic acid in the presence of excess diisopropylethylamine. The therapeutic gene used as an example in this protocol encodes the murine interleukin 12 (IL-12) subunits p35 and p40, each under the transcriptional control of a separate cytomegalovirus (CMV) promoter; the reporter plasmid contains the luciferase gene driven by a CMV promoter. The target cells are C-26 colon carcinoma cells.
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| Authors | Ram I Mahato, Zhaoyang Ye, Sung Wan Kim |
| Journal | Cold Spring Harbor protocols (Cold Spring Harb Protoc) Vol. 2009 Issue 10 Pg. pdb.prot5306 (Oct 2009) ISSN: 1559-6095 [Electronic] United States |
| PMID | 20147051 (Publication Type: Journal Article) |
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