Long-term expression after infection by the hybrid vector AdLTR-luc is from integrated transgene.
The novel adenoretroviral vector, AdLTR-luc, infects dividing and nondividing cells, and mediates long-term transgene expression(Zheng, C., Baum, B. J., Iadarola, M. J., and O'Connell, B. C., Nat. Biotech. 18, 176-180, 2000). To determine the source of this expression we examined two epithelial cell lines. One, HSG, permits E1(-) recombinant adenoviral replication, while the other, A5, does not. An HSG clone, that expressed luciferase stably for > 6 months, was obtained following infection at approximately 0.2 AdLTR-luc particles/cell. Southern and PCR analyses showed that luciferase cDNA present was integrated. A5 cells were infected with AdLTR-luc at approximately 1000 particles/cell, and colonies were obtained by limiting dilution. Eight clones showed stable luciferase activity for > 9 months. High molecular weight DNA extracts from clones were positive for genomic integration by Southern, PCR, and quantitative PCR analyses. Similar analyses of low molecular weight DNA extracts indicated the absence of intact extrachromosomal vector. These data demonstrate that long-term luciferase expression after infection by AdLTR-luc is derived from the integrated cDNA.
Zheng
Changyu
C
Gene Therapy and Therapeutics Branch, National Institutes of Health, Bethesda, Maryland 20892-1190, USA.
Baum
Bruce J
BJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D013997:Time Factors; D019076:Transgenes; D016662:Virus Integration
D018076:DNA, Complementary; D008156:Luciferases