Formation and use of water-soluble lipopolymer-pDNA and lipopeptide-pDNA complexes.
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.
Ram I
RI
Mahato
Zhaoyang
Z
Ye
Sung Wan
SW
Kim
D000818:Animals; D004247:DNA / Q000737:chemistry*; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry / Q000378:metabolism; D055666:Lipopeptides / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D008957:Models, Genetic; D010539:Permeability; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011479:Protamines / Q000737:chemistry; D014158:Transcription, Genetic; D014867:Water / Q000737:chemistry*
D055666:Lipopeptides; D011108:Polymers; D011479:Protamines; D014867:Water; D018664:Interleukin-12; D011094:Polyethyleneimine; D004247:DNA