PMID- 14984928
OWN - NLM
STAT- MEDLINE
DCOM- 20040604
LR  - 20220311
IS  - 0006-3002 (Print)
IS  - 0006-3002 (Linking)
VI  - 1676
IP  - 3
DP  - 2004 Feb 20
TI  - Efficient transfer of chromosome-based DNA constructs into mammalian cells.
PG  - 223-30
AB  - 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.
FAU - Oberle, Volker
AU  - Oberle V
AD  - Department of Membrane Cell Biology, Faculty of Medical Sciences, University of 
      Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
FAU - de Jong, Gary
AU  - de Jong G
FAU - Drayer, Jan I
AU  - Drayer JI
FAU - Hoekstra, Dick
AU  - Hoekstra D
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - Biochim Biophys Acta
JT  - Biochimica et biophysica acta
JID - 0217513
RN  - 0 (1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine)
RN  - 0 (Hanks Balanced Salt Solution)
RN  - 0 (Isotonic Solutions)
RN  - 0 (Phosphatidylethanolamines)
RN  - 0 (Pyridinium Compounds)
RN  - 0 (SAINT 2)
SB  - IM
MH  - Animals
MH  - Cells, Cultured
MH  - *Chromosomes, Artificial
MH  - Genetic Therapy/methods
MH  - Genetic Vectors/*pharmacology
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Isotonic Solutions
MH  - Particle Size
MH  - Phosphatidylethanolamines
MH  - Pyridinium Compounds
MH  - Temperature
MH  - Time Factors
MH  - Transfection/*methods
MH  - Ultrasonics
EDAT- 2004/02/27 05:00
MHDA- 2004/06/05 05:00
CRDT- 2004/02/27 05:00
PHST- 2003/07/02 00:00 [received]
PHST- 2003/12/15 00:00 [revised]
PHST- 2003/12/15 00:00 [accepted]
PHST- 2004/02/27 05:00 [pubmed]
PHST- 2004/06/05 05:00 [medline]
PHST- 2004/02/27 05:00 [entrez]
AID - S0167478103002896 [pii]
AID - 10.1016/j.bbaexp.2003.12.003 [doi]
PST - ppublish
SO  - Biochim Biophys Acta. 2004 Feb 20;1676(3):223-30. doi: 
      10.1016/j.bbaexp.2003.12.003.