Efficient transfer of chromosome-based DNA constructs into mammalian cells

• Published in: Biochimica et biophysica acta 2004-02, Vol.1676 (3), p.223-230
• Main Authors: Oberle, Volker, de Jong, Gary, Drayer, Jan I., Hoekstra, Dick
• Format: Article
• Language: English
• Subjects: Animals; artificial chromosomes; Cationic lipid; Cells, Cultured; Chromosomes, Artificial; DNA; Genetic Therapy - methods; Genetic Vectors - pharmacology; Humans; In Situ Hybridization, Fluorescence; Isotonic Solutions; Mammalian artificial chromosome; Particle Size; Phosphatidylethanolamines; Pyridinium Compounds; Temperature; Time Factors; Transfection - methods; Transfer; Ultrasonics; Ultrasound
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/j.bbaexp.2003.12.003

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 μm). 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.