Chromosomal engineering
Alistair Duncan
1
and Gyula Hadlaczky
2
Artificial chromosomes (ACs) are engineered chromosomes
with defined genetic contents that can function as non-
integrating vectors with large carrying capacity and stability.
The large carrying capacity allows the engineering of ACs with
multiple copies of the same transgene, gene complexes, and to
include regulatory elements necessary for the regulated
expression of transgene(s). Artificial chromosome based
systems are composed of AC engineered to harbor and
express gene(s) of interest and an appropriate recombination
system for ‘custom’ engineering of ACs. These systems have
the potential to become an efficient tool in diverse gene
technology applications such as cellular protein manufacturing,
transgenic animal production, and ultimately gene therapy.
Recent advances in artificial chromosome technologies outline
the value of these systems and justify the future research efforts
to overcome the obstacles in exploring their full capabilities.
Address
1
Chromos Molecular Systems Inc., 8081 Lougheed Highway, Burnaby,
BC, Canada V5A 1W9
2
Institute of Genetics, Biological Research Center, Hungarian Academy
of Sciences, H-6726 Szeged, Temesvari krt. 62, Hungary
Corresponding author: Hadlaczky, Gyula (hgy@brc.hu)
Current Opinion in Biotechnology 2007, 18:420–424
This review comes from a themed issue on
Expression technologies
Edited by Hansjoerg Hauser and Martin Fussenegger
Available online 30th October 2007
0958-1669/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2007.09.004
Introduction
Research on artificial chromosomes of higher eukaryotes
is now in its second decade. Four different strategies have
been developed for the construction of artificial chromo-
somes: (i) synthetic or ‘bottom up’ approach aiming the de
novo assembly of chromosomal components, (ii) ‘top
down’ method applying the in vivo telomere associated
fragmentation of existing chromosomes, (iii) engineering
of naturally occurring minichromosomes, and (iv) induced
de novo chromosome generation via targeted amplification
of specific chromosomal segments. Details of these
approaches including their evaluation have recently been
published [1
]. This review focuses on the recent progress
of technologies that turned the artificial chromosome
concept to reality. To date, two technologies, the ‘top
down’ and the induced de novo chromosome generation
approaches have been advanced to artificial chromosome
based systems (DHAC and the ACE systems), which
are ready for certain applications in biotechnology. These
systems are now offering efficient alternatives to produce
therapeutic proteins in bioreactors and in transgenic
organisms.
DHAC system
Artificial chromosomes of this system were produced by
sequential truncation of long (q) and short (p) arms of
human chromosome 21 (21DpHAC and 21DpqHAC). A
loxP site inserted into the ACs provides the insertion site
for the loading of transgene by the cre-recombinase-
mediated Cre/loxP system [2
]. Basic principles of the
DHAC system are summarized in Figure 1.
Inherent shortcomings of the cre-recombinase systems
are the presence of functional recognition sites in mam-
malian genomes that may lead to insertional mutagenesis
[3] and the low efficiency in most mammalian cells.
Truncation of chromosomes and efficient engineering
of DHACs usually require the transfer of chromosomes
into the homologous recombination proficient chicken
DT40 cells with either cell fusion or microcell-mediated
chromosome transfers [4], which are laborsome and
generally inefficient procedures [1
]. The competing
integration and excision activity of cre-recombinase
makes the multiple loading of transgenes unreliable.
However, these difficulties could be overcome with a
single engineered construct carrying multiple transgenes
and by applying appropriate quality control and selection
for clones with the desired engineered chromosome.
The artificial chromosome expression
system (ACE system)
The ACE system was built on the satellite DNA based
artificial chromosomes (SATACs) [5,6] that were gener-
ated by induced large-scale amplification of the short arm
of acrocentric chromosomes (centromeric/rDNA region).
These platform SATACs are composed predominantly of
amplified satellite and rDNA sequences and carry
multiple copies (>50) of co-amplified recombination
acceptor sites (attB) for multiple unidirectional loading
of expression cassettes by a modified l integrase (ACE
integrase) (Figure 2).
To fill additional acceptor sites, subsequent targeting(s)
can be performed either with the same gene or with
different construct(s). The number of loading rounds is
limited only by the number of selectable markers avail-
able. The ACE integrase can function autonomously in
mammalian cells and catalyzes site-specific integration at
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