Abstract
When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.
Highlights
Since the advent of human adenovirus (HAdV) vectors in the mid-1980’s, their use has expanded to address questions in almost all realms of applied and fundamental biology
Later Chartier et al developed an approach where HAdV genomes could be generated using homologous recombination (HR) in E. coli [4]. Using these approaches the adenovirus genome is released by restriction enzyme(s) at sites flanking the inverted terminal repeats (ITRs), and the DNA is transfected into cells to generate the vectors
We screened a handful of canine cell lines, but none was efficiently transduced by linear 30 kb DNA fragments
Summary
In the early 1990’s, recombinant vectors were generated via homologous recombination (HR) in cell lines [1]. To circumvent the generation and cloning in cell lines, and the time involved in growing and screening plaques, Ketner et al cloned HAdV genomes in plasmids using HR in S. cerevisiae [3]. Later Chartier et al developed an approach where HAdV genomes could be generated using HR in E. coli [4]. Using these approaches the adenovirus genome is released by restriction enzyme(s) at sites flanking the inverted terminal repeats (ITRs), and the DNA is transfected into cells to generate the vectors
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