Electric field-mediated gene transfer: characterization of DNA transfer and patterns of integration in lymphoid cells.
Southern analysis of individual transfectants generated by electroporation demonstrated a strong preference for the integration of DNA in low copy number even when electroporation was performed in the presence of increasing DNA concentrations. Although transfer of multiple DNA copies was detected at higher DNA concentrations (16 pmoles/ml or greater), the average gene copy number even at 36 pmoles DNA per ml, was only 13. Multiple gene copies were integrated at either a few chromosomal sites, or at a single site within individual transfectants. Restriction endonuclease cleavage data were consistent with a random orientation of molecules within a concatemer, suggesting that the concatemer may have risen via end-to-end ligation of linear molecules, rather than by homologous recombination. Integration of exogenous DNA into the host chromosome occurred preferentially at the ends of the linear molecule. Although the linearization site was lost upon integration, endonuclease sites as close as 18 bp from the linearization site were retained. These data, as well as direct restriction mapping of the transferred genes, indicate that DNA transfer and integration occur without DNA rearrangement. Taken together, these results suggest that electroporation may offer some unique advantages for the transfer of eukaryotic genes.
Toneguzzo
F
F
EG & G Biomolecular, Natick, MA 01760.
Keating
A
A
Glynn
S
S
McDonald
K
K
D000818:Animals; D001402:B-Lymphocytes; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000235:genetics; D005821:Genetic Techniques; D008223:Lymphoma; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014162:Transfection
D004273:DNA, Neoplasm; D004262:DNA Restriction Enzymes