Question

Describe the production of Adenovirus vector in detail.

Answer 1

Answer=

production of Adenovirus vector =

Adenoviruses are nonenveloped DNA viruses consisting of a linear,double stranded DNA genome of approximately 30-40 kbp.

there are number of strategies have been developed to construct Ad vectors carrying a foreign gene insert.

Traditionally there are 2 methods:

1] In vitro ligation method - involving the ligation of a DNA fragment obtained by restriction restriction digesting of a plasmid carrying the foreign gene insert flanked by Ad sequences with a DNA fragment representing the rest of Ad genome.

2] Consists of homologous recombination in permissive cell lines between two plasmids the shuttle plasmid is carrying the foreign gane insert flanked by Ad sequences for site -specific insertion and the genomic plasmid carrying almost entire Ad genome.

new strategy= One strategy is based on the highly efficient homologous recombination machinary of E.coli to generate Ad vectors.

homologous recombination between a linearized or intact plasmid containing almost an entire Ad genome & a shuttle plasmid containing an exogenous expression cassette flanked by homogenous sequences from the site of insertion in the Ad genome generates an infectious clone with a modification or insertion in the desired region.

homologous recombination in yeast involves recombination between Ad DNA and yeast artificial chromosome (YAC) vector containing sequences from the left & right termini of Ad genome,resulting in the generation of a yeast artificial chromosome containing an infectious copy of Ad genome.

transfection of the excised Ad genome into appropriate cells results in generation of infectious virions.