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.
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Volker
V
Oberle
Gary
G
de Jong
Jan I
JI
Drayer
Dick
D
Hoekstra
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