Electric field-mediated gene transfer: characterization of DNA transfer and patterns of integration in lymphoid cells.
AUTOR(ES)
Toneguzzo, F
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=336782Documentos Relacionados
- Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.
- Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field-mediated DNA transfer.
- DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid.
- Human gene transfer: characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man.
- DNA-mediated gene transfer of a circular plasmid into murine cells.