Optimized cationic lipid-based gene delivery reagents for use in developing vertebrate embryos.
We have used cationic lipid-based transfection reagents for ectopic gene expression experiments in developing vertebrate embryos. Lipofectamine, Lipofectamine 2000, and Lipofectamine enhanced with a disulfide linked pegylated lipid (mPEG-SS-DOPE) were initially tested and optimized in cell culture. Two reagent formulations, 1:4 (DNA:Lipofectamine 2000) Lipofectamine 2000, and 7.5% pegylated Lipofectamine, produced the highest levels of gene expression in vitro. Those formulations, containing the enhance green fluorescent protein reporter gene, were microinjected into intact vertebrate embryos -- systemically through the vasculature and locally into selected tissues -- to assess in vivo transfection efficiency. Whereas both formulations are capable of transfecting cells in developing embryos in vivo, greater transfection efficiencies in a broader range of tissue types were obtained with the pegylated Lipofectamine formulation. We conclude that in developing vertebrate embryos, optimized cationic lipid-based reagents are capable of producing significant levels of ectopic gene expression and can be used as alternatives to electroporation and viral-mediated gene delivery.
Decastro
Michael
M
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Saijoh
Yukio
Y
Schoenwolf
Gary C
GC
D000818:Animals; D002412:Cations / Q000737:chemistry; D002478:Cells, Cultured; D002642:Chick Embryo; D004337:Drug Carriers / Q000737:chemistry* / Q000494:pharmacology*; D008055:Lipids / Q000737:chemistry* / Q000494:pharmacology*; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry; D014162:Transfection / Q000379:methods*; D014714:Vertebrates / Q000196:embryology* / Q000378:metabolism*
D002412:Cations; D004337:Drug Carriers; D008055:Lipids; D011092:Polyethylene Glycols