PMID- 10476208
OWN - NLM
STAT- MEDLINE
DCOM- 19990923
LR  - 20131121
IS  - 0969-7128 (Print)
IS  - 0969-7128 (Linking)
VI  - 6
IP  - 4
DP  - 1999 Apr
TI  - Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene 
      transfer.
PG  - 482-97
AB  - Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting 
      hurdles to gene transfer, utilizing a nonviral delivery system such as 
      polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA 
      has to be released into the cytosol before its nuclear entry can occur. However, 
      the stability of plasmid DNA in the cytoplasm, that may play a determinant role 
      in the transfection efficiency, is not known. The turnover of plasmid DNA, 
      delivered by microinjection into the cytosol, was determined by fluorescence in 
      situ hybridization (FISH) and quantitative single-cell fluorescence video-image 
      analysis. Both single- and double-stranded circular plasmid DNA disappeared with 
      an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, 
      while the amount of co-injected dextran (MW 70,000) remained unaltered. We 
      propose that cytosolic nuclease(s) are responsible for the rapid-degradation of 
      plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell 
      division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of 
      microinjected plasmid DNA was inhibited in cytosol-depleted cells or following 
      the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent 
      elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl 
      transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the 
      fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by 
      selective permeabilization of the plasma membrane, exhibits divalent 
      cation-dependent, thermolabile nuclease activity, determined by Southern blotting 
      and 32P-release from end-labeled DNA. Collectively, these findings suggest that 
      the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may 
      constitute a previously unrecognized impediment for DNA translocation into the 
      nucleus and a possible target to enhance the efficiency of gene delivery.
FAU - Lechardeur, D
AU  - Lechardeur D
AD  - Program in Cell and Lung Biology and Lung Gene Therapy, Hospital for Sick 
      Children, Toronto, Ontario, Canada.
FAU - Sohn, K J
AU  - Sohn KJ
FAU - Haardt, M
AU  - Haardt M
FAU - Joshi, P B
AU  - Joshi PB
FAU - Monck, M
AU  - Monck M
FAU - Graham, R W
AU  - Graham RW
FAU - Beatty, B
AU  - Beatty B
FAU - Squire, J
AU  - Squire J
FAU - O'Brodovich, H
AU  - O'Brodovich H
FAU - Lukacs, G L
AU  - Lukacs GL
LA  - eng
GR  - N01-HD-7-3263/HD/NICHD NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Gene Ther
JT  - Gene therapy
JID - 9421525
RN  - EC 3.1.- (Deoxyribonucleases)
RN  - KOO5CM684H (Digitonin)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Blotting, Southern
MH  - Calcium/metabolism
MH  - Cytosol/*metabolism
MH  - Deoxyribonucleases/metabolism
MH  - Digitonin/metabolism
MH  - *Gene Transfer Techniques
MH  - HeLa Cells
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Microinjections
MH  - Microscopy, Fluorescence
MH  - Plasmids/*metabolism
MH  - *Transfection
EDAT- 1999/09/07 00:00
MHDA- 1999/09/07 00:01
CRDT- 1999/09/07 00:00
PHST- 1999/09/07 00:00 [pubmed]
PHST- 1999/09/07 00:01 [medline]
PHST- 1999/09/07 00:00 [entrez]
AID - 10.1038/sj.gt.3300867 [doi]
PST - ppublish
SO  - Gene Ther. 1999 Apr;6(4):482-97. doi: 10.1038/sj.gt.3300867.