Efficient transfer of chromosome-based DNA constructs into mammalian cells.
Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.
Oberle
Volker
V
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
de Jong
Gary
G
Drayer
Jan I
JI
Hoekstra
Dick
D
D000818:Animals; D002478:Cells, Cultured; D022201:Chromosomes, Artificial; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000494:pharmacology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007552:Isotonic Solutions; D010316:Particle Size; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; D013696:Temperature; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; C072670:Hanks Balanced Salt Solution; D007552:Isotonic Solutions; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; C414306:SAINT 2