Biology Reference
In-Depth Information
Chapter 11
Engineering Adenovirus Genome by Bacterial Artifi cial
Chromosome (BAC) Technology
Zsolt Ruzsics , Frederic Lemnitzer , and Christian Thirion
Abstract
Bacterial artifi cial chromosomes (BACs) are recombinant DNA molecules designed for propagation of
large and instable foreign DNA fragment in
Escherichia coli
. BACs are used in genetics of large DNA
viruses such as herpes and baculoviruses for propagation and manipulation of complex genomic regions or
even entire viral genomes in one piece. Viral genomes in BACs are ready for the advanced tools of
E. coli
genetics. These techniques based on homologous or site-specifi c recombination allow engineering of vir-
tually any kind of genetic changes. In the recent years, BAC technology was also adapted to manipulation
of adenovirus genomes and became an effective alternative to traditional genetic engineering of recombi-
nant adenoviruses.
Key words
Bacterial artifi cial chromosome, Recombineering, Homologous recombination in
E. coli
,
Red recombinases, Flp/FRT recombination, Recombinant adenoviruses
1
Introduction
Vectors based on human adenoviruses (Ads) of species C, in par-
ticular the best-characterized serotype Ad5, appeared as extremely
useful vehicles for gene therapy and vaccination. In the past few
years, however, there has been growing interest in studying Ad
vectors derived from species other than C. This was partially ham-
pered by the lack of convenient systems for maintenance and
manipulation of their recombinant genomes. This motivated us to
develop a bacterial artifi cial chromosome (BAC)-based technology
for Ads [
1
], which provides an unparalleled access to genome of
large DNA viruses utilizing the advanced methodology of bacterial
genetics [
2
].
In adenovirus genetics, BAC technology is especially useful for
the establishment of reverse genetics systems when the Ad genomes
under study are unstable in traditional high-copy vector systems
(i.e., Ad19a, Ad3) [
1
,
3
]. In addition, the BAC technology allows
construction of recombinant genomes using a little aliquot of Ad
