A new and efficient method for gene transfer into mouse FM3A cells using metaphase chromosomes by electroporation.
We introduced chromosome-mediated genes into mouse thymidine kinase-deficient FM3A (FM3Atk-) cells, by electroporation. The effects of some parameters on the electric shock-mediated transfection of FM3Atk- cells were investigated. Gene transfer of mouse L929 metaphase chromosome DNA into FM3Atk- resulted in a maximum frequency of (3.0 +/- 0.3) x 10(-5) at a cell density of 2.0 x 10(8)/ml and chromosome dosage of 5.0 x 10(7) cell equivalents/ml in a buffer containing 0.25 M mannitol, 0.5 mM MgCl2, 0.1mM CaCl2, and 1 mM Tris-HCl (pH 7.1). The highest yield of the transformants was obtained at an electric field strength of 1 kV/cm and a capacitance of 35 microF, with a single exponentially decaying pulse at 0 degrees C was optimal for post-shock incubation after electroporation. The tk gene was detected in the transformants by in situ hybridization analysis.
Ohse
M
M
Department of Applied Physics and Chemistry, Faculty of Engineering, Fukui University of Technology, Japan.
Tsuchida
K
K
Tomita
H
H
Taketo
A
A
Kimoto
H
H
Kusaoke
H
H
D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000502:physiology* / Q000648:ultrastructure; D003470:Culture Media; D004247:DNA / Q000378:metabolism; D018274:Electroporation; D018014:Gene Transfer Techniques; D017403:In Situ Hybridization; D008677:Metaphase / Q000502:physiology*; D051379:Mice; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D004247:DNA; D013937:Thymidine Kinase