重组腺病毒理论PPT.ppt

gutless or gutted Ad: devoid of all coding viral regions helper-dependent adenoviruses: because of the need of a helper adenovirus that carries all coding regions high-capacity adenoviruses: because they can accommodate up to 36 kb of DNA only keeps the 5’ and 3’ inverted terminal repeats (ITRs) and the packaging signal from the wild-type adenovirus. Third-generation adenoviral vectors Generation of gutless adenovirus using the Cre/loxP system. Gutless and helper genomes are cotransfected in permissive 293 Cre-expressing cells, where both genomes are amplified and viral proteins produced. Then, packaging signal of the helper’s genome is excised by Cre recombinase, preventing its packaging into the viral capsid, while gutless genome is still packageable. Efficiency of the excision process allows 90–99.9% purity of the gutless vector. Design and Construction of Adenoviral Vectors In order to provide additional cloning space in the vector, the E3 region, which is not necessary for viral replication in culture, is also commonly deleted. homologous recombination in an E1-complementing human cell line between two DNA molecules: one carrying sequences mapping to the left end of the Ad genome and the gene of interest; one carrying the Ad genome with the left end deleted but retaining some sequences that partially overlap the 3 end of the first molecule Homologous recombination inefficiency of homologous recombination in mammalian cells; the need for purification of individual viral plaques, which means it is both labor-intensive and time-consuming; if no recombinants are generated, the researcher is unable to determine whether the problem is technical or biological. Problems exploits the highly efficient homologous recombination machinery in bacteria to generate a recombinant Ad vector by homologous recombination in Escherichia coli between a large plasmid containing most of the Ad genome and a small shuttle plasmid containing the expression cassette flanked by seque