Idioma:

Metabolic instability of plasmid DNA in the cytosol : a potential barrier to gene transfer

LECHARDEUR, D ; SOHN, K.-J ; HAARDT, M ; JOSHI, P. B ; MONCK, M ; GRAHAM, R. W ; BEATTY, B ; SQUIRE, J ; O'BRODOVICH, H ; LUKACS, G. L

Gene therapy, 1999-04, Vol.6 (4), p.482-497 [Periódico revisado por pares]

Basingstoke: Nature Publishing Group

Texto completo disponível

Citações Citado por

Enviar para

Título:
Metabolic instability of plasmid DNA in the cytosol : a potential barrier to gene transfer
Autor: LECHARDEUR, D ; SOHN, K.-J ; HAARDT, M ; JOSHI, P. B ; MONCK, M ; GRAHAM, R. W ; BEATTY, B ; SQUIRE, J ; O'BRODOVICH, H ; LUKACS, G. L
Assuntos: Apoptosis ; Biological and medical sciences ; Biotechnology ; Blotting, Southern ; Calcium - metabolism ; Cell division ; Cytoplasm ; Cytosol ; Cytosol - metabolism ; Deoxyribonucleases - metabolism ; Deoxyribonucleic acid ; Dextran ; Digitonin - metabolism ; DNA ; DNA fragmentation ; DNA nucleotidylexotransferase ; Endocytosis ; Fluorescence in situ hybridization ; Fundamental and applied biological sciences. Psychology ; Gene therapy ; Gene transfer ; Gene Transfer Techniques ; Health. Pharmaceutical industry ; HeLa Cells ; Humans ; Hybridization ; Image processing ; In Situ Hybridization, Fluorescence ; Industrial applications and implications. Economical aspects ; Internalization ; Life Sciences ; Metabolism ; Microinjection ; Microinjections ; Microscopy, Fluorescence ; Nuclease ; Other ; Phospholipids ; Plasmids - metabolism ; Southern blotting ; Transfection
É parte de: Gene therapy, 1999-04, Vol.6 (4), p.482-497
Notas: ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ObjectType-Article-1
ObjectType-Feature-2
Descrição: Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.
Editor: Basingstoke: Nature Publishing Group
Idioma: Inglês

Links

Voltar para lista de resultados

Realização: Logos de Redes Sociais:

Metabolic instability of plasmid DNA in the cytosol : a potential barrier to gene transfer

LECHARDEUR, D ; SOHN, K.-J ; HAARDT, M ; JOSHI, P. B ; MONCK, M ; GRAHAM, R. W ; BEATTY, B ; SQUIRE, J ; O'BRODOVICH, H ; LUKACS, G. L

Basingstoke: Nature Publishing Group

Buscando em bases de dados remotas. Favor aguardar.