Abstract
This chapter includes all the protocols required to construct and test EAMs with any transgene of interest. A discussion of the main features of the different types of EAMs and of the modified protocols, developed for analysis of EAM-mediated expression of transgenes in the retina has been provided. The construction of EAMs depends on two seminal observations regarding the nature of the origin of replication and encapsidation signals of adenovirus (Ad). EAMs were constructed to address the issue of the strong host immune response observed during clinical trials using first-generation Ad vectors. These are vectors that have the adenoviral region E1 deleted and replaced with the transgene of interest. Also included in the first-generation vectors are usually those Ad vectors with deletions in region E3. First-generation Ad vectors are propagated in human embryonic kidney 293 cells, which provide the E1 proteins in trans. To reduce the number of viral proteins being expressed in infected cells, second-generation viral vectors have been constructed, usually with deletions in genes coding for the viral replication machinery, further crippling these viruses in vivo . However, it is unclear if such viruses result in prolonged expression when compared with first-generation Ad vectors.
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