Formation and use of water-soluble lipopolymer-pDNA and lipopeptide-pDNA complexes

Ram I. Mahato, Zhaoyang Ye, Sung Wan Kim

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalCold Spring Harbor Protocols
Volume4
Issue number10
DOIs
StatePublished - 2009

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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