Efficient introduction of a gene into hematopoietic cells in S-phase by electroporation.
Cells of the hematopoietic cell line K562 were synchronized by three different methods: single aphidicolin treatment, thymidine treatment followed by hydroxyurea exposure, and double hydroxyurea treatment. The synchronized cells were transfected via electroporation with plasmid pMoZtk, which contains the beta-galactosidase gene, using a square wave pulse immediately after synchronization or at various time points during culture. Simultaneously, synchronized cells were fluorescence-activated cell sorter (FACS) analyzed to determine their stage in the cell cycle using double staining with bromodeoxyuridine (BrdU) and propidium iodide. Highly efficient introduction of pMoZtk was observed for the cell fraction, which predominantly consisted of the cells in S-phase. These results suggest that by increasing the proportion of cells in S-phase, the efficiency of gene transfer into hematopoietic cells such as hematopoietic stem cells can be improved.
Takahashi
M
M
First Department of Internal Medicine, Niigata University, School of Medicine, Asahimachi, Japan.
Furukawa
T
T
Nikkuni
K
K
Aoki
A
A
Nomoto
N
N
Koike
T
T
Moriyama
Y
Y
Shinada
S
S
Shibata
A
A
D016590:Aphidicolin; D001973:Bromodeoxyuridine; D002460:Cell Line; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors; D004224:Diterpenes / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D010957:Plasmids; D011419:Propidium; D016196:S Phase / Q000187:drug effects / Q000502:physiology*; D013936:Thymidine / Q000494:pharmacology; D014162:Transfection / Q000235:genetics / Q000502:physiology*; D001616:beta-Galactosidase / Q000235:genetics*
D004224:Diterpenes; D011419:Propidium; D016590:Aphidicolin; D004257:DNA Polymerase II; D001616:beta-Galactosidase; D001973:Bromodeoxyuridine; D013936:Thymidine; D006918:Hydroxyurea