Electric field-mediated gene transfer into K562 cells: optimization of parameters affecting efficiency.
Since hemopoietic cells are refractory to transfection by conventional chemical means, we have developed a reliable and efficient gene transfer system for K562 cells which uses electric field-mediated gene transfer (EFMGT). EFMGT involves the exposure of cells in suspension to an electric field which transiently allows the entry of DNA into the cell and its subsequent integration and expression. Plasmids bearing the neo gene were used to identify and select transfected clonogenic cells manifested by geneticin resistance in semisolid medium. Transfection efficiency is significantly affected by the following variables: voltage, capacitance, time constant, number of pulses, buffer type and temperature, DNA concentration, configuration, and promoter type. Cell cycle status also appears to be critical as shown in studies employing aphidicolin synchronization. Using optimal conditions, we have consistently achieved a transfection efficiency of 0.3-0.4% of clonogenic cells per microgram DNA. Stability of neo gene expression was also demonstrated after 4 months in nonselective culture conditions. This level of efficiency compares favorably with other reports of gene transfer into human hemopoietic progenitor cells.
Croaker
G M
GM
Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
Wass
E J
EJ
Iland
H J
HJ
D001752:Blast Crisis / Q000235:genetics*; D002021:Buffers; D002453:Cell Cycle; D004247:DNA / Q000235:genetics; D006801:Humans; D007942:Leukemia, Experimental / Q000235:genetics* / Q000473:pathology; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000473:pathology; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D002021:Buffers; D004247:DNA