Optimization of a Peptide/Non-cationic Lipid Gene Delivery System for Effective Microinjection into Chicken Embryo in Vivo

Abstract

Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.

Recent Trends of Polymer Mediated Liposomal Gene Delivery System

“…A parallel approach also resulted in a functional complex that can be used for targeted gene delivery using amphipathic peptide derivatives as DNA packing agents and a bulk of neutral helper lipid to prepare DNA complexes by the detergent dialysis method [150]. Lately, numerous aspects related to lipid composition, the presence of shielding PEG-lipid conjugates, and the nature of chemical bonding that contributes to the biodegradability of the PEG-lipid conjugates in cells have been thoroughly studied [151, 152]. Such ideas are significantly not the same from the original lipid-DNA and/or polymer-DNA complexes and certainly deserve further exploration, particularly in the area of in vivo targeted gene delivery.…”

Optimized cationic lipid‐based gene delivery reagents for use in developing vertebrate embryos

“…Polyethylene glycol was conjugated to dioleoyl phosphatidylethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL) with the bifunctional crosslinker dithiobis(succinimidyl propionate) (DSP; Pierce, Rockford, IL), as previously described (Kirpotin et al, 1996;Longmuir et al, 2001). Briefly, methoxypolyethylene glycol amine, mPEG-NH 2 (Nektar Therapeutics, Huntsville, AL), dissolved in dimeth-ylformamide (DMF) was slowly added dropwise to a fivefold molar excess of DSP dissolved in DMF, while stirring vigorously.…”

Multiphoton excitation spectra in biological samples

“…Transfection of GFP Expressing Plasmid ͑gWIZ, GTS, San Diego, California͒ using Lipomer transfection reagent was performed essentially as previously described. 13 The incubation media was replaced with media containing 2 l/1 ml of transfection reagent and cells were incubated for 12 to 14 h. H2B-CFP, H2B-GFP, and H2B-YFP fusion proteins were introduced into 3T3 cells via viral infection as previously described in Ref. 1.…”