Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus.
Anticancer Res
; 22(6A): 3325-30, 2002.
Article
em En
| MEDLINE
| ID: mdl-12530082
ABSTRACT
BACKGROUND:
Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines. MATERIALS ANDMETHODS:
We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice.RESULTS:
Stable HT-29 clones with > 97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression.CONCLUSION:
This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transfecção
/
Regiões Promotoras Genéticas
/
Dependovirus
/
Células HT29
/
Fator 1 de Elongação de Peptídeos
/
Citomegalovirus
Tipo de estudo:
Risk_factors_studies
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article