PMID- 11318114
OWN - NLM
STAT- MEDLINE
DCOM- 20010809
LR  - 20131121
IS  - 1099-498X (Print)
IS  - 1099-498X (Linking)
VI  - 3
IP  - 2
DP  - 2001 Mar-Apr
TI  - Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of 
      injected plasmids.
PG  - 153-64
AB  - BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in 
      gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a 
      major limiting step in gene transfer using non-viral vectors. It is commonly 
      thought that, in the cytosol, cDNA has to be released from its vector before 
      importation to the nucleus. The stability of naked cDNA in the cytoplasm is not 
      well established. METHODS: cDNA plasmids, either free or complexed with 
      poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian 
      cells and their turnover was assessed by fluorescence in situ hybridization 
      (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. 
      RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when 
      injected into the cytosol. Fading was not observed when naked cDNA plasmids were 
      injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction 
      isolated from mammalian cells reproduced cDNA degradation as observed in 
      microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic 
      acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease 
      activity was optimal at physiological pH and physiological Ca2+ concentration. By 
      contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA 
      complexation with PEI or addition of oligonucleotides prevented in vitro cDNA 
      degradation. CONCLUSION: Altogether, these experiments suggest that cDNA 
      digestion by cytosolic nucleases occur when the decomplexed transgene is present 
      in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus 
      during transfection with synthetic vectors may result from rapid digestion of 
      naked cDNA by a Ca2+-sensitive cytosolic nuclease.
FAU - Pollard, H
AU  - Pollard H
AD  - INSERM U533, Laboratoire de Physiopathologie, H pital H tel-Dieu, Nantes, France.
FAU - Toumaniantz, G
AU  - Toumaniantz G
FAU - Amos, J L
AU  - Amos JL
FAU - Avet-Loiseau, H
AU  - Avet-Loiseau H
FAU - Guihard, G
AU  - Guihard G
FAU - Behr, J P
AU  - Behr JP
FAU - Escande, D
AU  - Escande D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Gene Med
JT  - The journal of gene medicine
JID - 9815764
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Complementary)
RN  - 0 (Enzymes)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - Calcium/*metabolism
MH  - Cell Line
MH  - Cell Nucleus/*metabolism
MH  - Cytosol/*enzymology
MH  - DNA Primers
MH  - DNA, Complementary
MH  - Enzymes/metabolism
MH  - *Gene Transfer Techniques
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Plasmids/*administration & dosage
MH  - Polymerase Chain Reaction
EDAT- 2001/04/25 10:00
MHDA- 2001/08/10 10:01
CRDT- 2001/04/25 10:00
PHST- 2001/04/25 10:00 [pubmed]
PHST- 2001/08/10 10:01 [medline]
PHST- 2001/04/25 10:00 [entrez]
AID - 10.1002/jgm.160 [doi]
PST - ppublish
SO  - J Gene Med. 2001 Mar-Apr;3(2):153-64. doi: 10.1002/jgm.160.