Biomedical Engineering Reference
In-Depth Information
If we add the complexity and participation of cell nucleus compartments and their associ-
ated chromatin structures, it becomes clear that a lot of effort in experimental design and
development of new tools is needed for manipulating the genome [9, 11].
7.1.1 Artificial chromosomes and transgenesis
In the course of the last two decades, remarkable efforts have been made on the devel-
opment of new strategies for gene expression that incorporate large genomic sequences.
This is mainly because there is more and more evidence showing that genes require a
sophisticated set of proximal (promoters) and distal regulatory elements to achieve relevant
expression [12, 13]. Therefore, the use of artificial chromosomes from yeast and bacteria
has turned out to be an attractive alternative, not only to recapitulate endogenous gene
expression patterns, but also, even more interestingly, to manipulate large genomic regions
by taking advantage of homologous recombination strategies [14-16]. Thus, transgenesis
with artificial chromosomes has proven to be useful not only in the study of a variety of
regulatory and developmental processes, but also for biomedical and biotechnological appli-
cations [15]. Two classes of artificial chromosome vector types are commonly used with
large cloning capacities: the yeast artificial chromosomes (YACs) and the bacteria-derived
bacterial artificial chromosomes (BACs) or P1-artificial chromosomes (PACs). YAC and
BAC/PAC vectors permit the incorporation of genomic inserts ranging from 100 kb to more
than 1Mb. The insertion of such large genomic sequences facilitates inclusion of all the
regulatory sequences needed for gene expression. Furthermore, the incorporation of all the
elements required for gene expression certainly contributes towards ensuring positional inde-
pendence, copy-number dependence and optimal levels of transgene expression. In addition,
the most attractive and useful feature of artificial chromosomes is the apparent unlimited
capacity to generate a large variety of modifications that can be incorporated into such vec-
tors, including target disruption of specific sequences, inversion or even insertions [17-19].
7.1.2 Gene transfer and expression problems
Gene transfer expression faces two obstacles: the first consists of frequent variability in
transgene expression levels and the second, which is a less-studied phenomenon, is the
progressive extinction of expression or silencing of the transgenes. In both cases, the main
component responsible for such effects, also known as chromatin position effects, is chro-
matin structure [1, 2].
7.1.3 Position effects and chromatin
There are two types of position effect: chromatin position effects caused by different inte-
gration sites and position effect variegation induced by a rearrangement and subsequent
silencing of an active gene, frequently due to its inactivation because of its proximity
to heterochromatin [20-22]. Generally, position effect variegation has been defined as a
stochastic and heritable silencing of gene expression. Such an effect is particularly accen-
tuated when the integration of the transgene occurs close to heterochromatin, where the
silencing pressure is even stronger.
On the other hand, chromatin position effects are basically considered to be the variability
in gene expression due to random insertion of each transgene in diverse chromatin envi-
ronments in the genome. As a consequence, studies associated with chromatin remodeling
