Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells.
Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event.
Zheng
Changyu
C
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-1190, USA.
O'Connell
Brian C
BC
Baum
Bruce J
BJ
D000256:Adenoviridae / Q000235:genetics*; D017498:Adenovirus E1 Proteins / Q000235:genetics*; D002460:Cell Line; D004847:Epithelial Cells / Q000821:virology; D017353:Gene Deletion; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008156:Luciferases; D008979:Moloney murine leukemia virus / Q000235:genetics*; D020079:Terminal Repeat Sequences; D019076:Transgenes; D016662:Virus Integration / Q000235:genetics*
D017498:Adenovirus E1 Proteins; D008156:Luciferases