Rapid transport of plasmid DNA into the nucleolus via actin depolymerization using the HVJ envelope vector.
BACKGROUND
Although nuclear transport of therapeutic genes is an essential requirement of human gene therapy, factors required for nuclear entry of DNA remain to be elucidated. Non-viral vector systems have led to numerous improvements in the efficiency of delivery of exogenous DNA into cells. However, nuclear transport of plasmid is difficult to achieve.


METHODS
We examined nuclear translocation efficiency of Cy3-labeled plasmid DNA (Cy3-pDNA) delivered by the hemagglutinating virus of Japan envelope (HVJ-E) vector, Lipofectamine or microinjection. We also examined the effect of actin depolymerization on nuclear transport of Cy3-pDNA.


RESULTS
Cy3-pDNA reached the nucleus, particularly in the nucleolus, in 30 min after fusion-mediated delivery using the HVJ-E vector, while the DNA was retained in the cytoplasm during the observed period after the delivery by cationic liposomes. HVJ-E treatment transiently depolymerized actin filaments, and acceleration of nucleolar entry of microinjected DNA was achieved when treated with either empty HVJ-E or cytochalasin D, an inhibitor of actin depolymerization, prior to microinjection.


CONCLUSIONS
These results suggest that plasmid DNA can be transported rapidly from the cytoplasm to the nucleolus when actin filaments are depolymerized. Thus, the HVJ-E vector can accelerate the transport of DNA to the nucleolus by actin depolymerization.
Suvanasuthi
Saroj
S
Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Tamai
Katsuto
K
Kaneda
Yasufumi
Y
D000199:Actins / Q000378:metabolism*; D001692:Biological Transport; D002232:Carbocyanines / Q000378:metabolism; D002466:Cell Nucleolus / Q000378:metabolism*; D002478:Cells, Cultured; D004396:Coloring Agents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005456:Fluorescent Dyes; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008845:Microinjections; D010957:Plasmids / Q000235:genetics*; D029082:Sendai virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics
D000199:Actins; D002232:Carbocyanines; D004396:Coloring Agents; D005456:Fluorescent Dyes; D014759:Viral Envelope Proteins; C089187:cyanine dye 3; D004247:DNA