Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus.
BACKGROUND
Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines.


METHODS
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.


CONCLUSIONS
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.
Teschendorf
Christian
C
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, P.O. Box 100266, JHMHSC, Gainesville, FL 32610, USA. Christian.Teschendorf@ruhr-uni-bochum.de
Warrington
Kenneth H
KH
Siemann
Dietmar W
DW
Muzyczka
Nicholas
N
D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019073:HT29 Cells / Q000473:pathology / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D049452:Green Fluorescent Proteins