Optimization of electroporation for transfection of human fibroblast cell lines with origin-defective SV40 DNA: development of human transformed fibroblast cell lines with mucopolysaccharidoses (I-VII)
To simplify the process of transfection of human fibroblasts and to acquire a suitable number of transformants, we investigated experimental conditions of electric pulse-induced transfection of human fibroblasts using origin-defective simian virus 40 DNA (SV40 (ori-) DNA). Voltage, pulse duration, number of pulses and the concentration of SV40 (ori-) DNA led to the formation of 10 to 30 foci/25 cm2 6 weeks after transfection, using 2 to 3 x 10(6) cells and a square wave pulse generator. Optimal condition was determined to be 2 or 3 pulses at a voltage of 1500 to 2000 V/0.4 cm with 30 microseconds pulse width, using 2 micrograms of linearized SV40 (ori-) DNA. With this approach we developed human transformed fibroblasts cell lines with all types of mucopolysaccharidoses. The transformed fibroblasts grew rapidly and the saturation density exceeded that of the parental cells. All the transformed cell clones expressed T antigen, and deficiency in specific enzymes was conserved. A point mutation which occurred in the human beta-glucuronidase gene in a patient with mucopolysaccharidosis type VII was also conserved.
Okamoto
H
H
Department of Pediatrics, Gifu University School of Medicine, Japan.
Sukegawa
K
K
Tomatsu
S
S
Suzuki
Y
Y
Orii
T
T
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis; D001483:Base Sequence; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000235:genetics; D004560:Electricity; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009083:Mucopolysaccharidoses / Q000201:enzymology / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral