TY - JOUR
T1 - A novel method for generating and screening peptides and libraries displayed on adenovirus fiber
AU - Lupold, Shawn E.
AU - Kudrolli, Tarana A.
AU - Chowdhury, Wasim H.
AU - Wu, Ping
AU - Rodriguez, Ronald
N1 - Funding Information:
We thank Victor W. van Beusechem (VU University Medical Center, Amsterdam) for providing the 911-S11 cell line, Stephen Langer (University of Colorado, Boulder) for providing the 293cre57 cell line and for technical advice, Drew Pardoll, Sara Pai and David Johns (Johns Hopkins University School of Medicine) for providing parent modified Fiber vectors and FBJ cell line, respectfully, and Bert Vogelstein for providing pAdEasy-1, pAdTrack, pAdTrack-CMV, BJ5183 and AdEasier cells. This article was funded by National Institutes of Health (2P50CA58236-09A1), (1R01CA121153-01A2 to R.R.); the Robert & Donna Tompkins Foundation, an award from David H. Koch through the Prostate Cancer Foundation, and the Department of Defense Prostate Cancer Research Program under Award Number (DAMD17-03-2-0033), which is managed by the U.S. Army Medical Research and Material Command. Funding to pay the Open Access publication charges for this article was provided by the Prostate Cancer Foundation.
PY - 2007/11
Y1 - 2007/11
N2 - Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.
AB - Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Cre-lox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The 'acceptor' vector does not contain the fiber gene, and therefore does not propagate until it has received a 'donor' fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.
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U2 - 10.1093/nar/gkm914
DO - 10.1093/nar/gkm914
M3 - Article
C2 - 17965092
AN - SCOPUS:36749092228
SN - 0305-1048
VL - 35
JO - Nucleic acids research
JF - Nucleic acids research
IS - 20
M1 - e138
ER -