Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations.
We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.
Tatsuka
M
M
Institute of Molecular and Cellular Biology, Kyoto Pharmaceutical University, Japan.
Yamagishi
N
N
Wada
M
M
Mitsui
H
H
Ota
T
T
Odashima
S
S
D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D008322:Mammals; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D015203:Reproducibility of Results; D014162:Transfection