Biomedical Engineering Reference
In-Depth Information
regulation, post-translational events, chromatin composition, recombinant protein production
and gene therapy, among others. One particular fact to be taken into consideration is that
there is no general methodology for gene transfer and, therefore, gene expression studies.
Each cell type and organism needs prior careful characterization to ensure optimal transfer
conditions to reach the highest efficiencies and reproducibility. In the great majority of
experimental biological systems we are constantly confronted with two variables. The first
one has to do with the great inconsistency in the expression levels of transgenes and the
second with a less-studied phenomenon, namely the progressive extinction of transgene
expression, which is present in the great majority of the cases [1, 2]. In addition, a less-clear
general phenomenon has to do with multiple copies of the same transgene which, once
integrated into the host genome, induces a phenomenon called co-suppression in plants,
which causes gene expression silencing when multiple copies of transgenes are integrated
in tandem [3].
The lack of accessible
in vivo
systems compels us to search for alternatives to gene
expression in eukaryotic cells. Over the years a large list of gene transfer applications has
arisen, and the constant appearance of new methodologies has made it more accessible
and reproducible for research scientists [4]. One of the most common applications for gene
transfer is the study of gene expression patterns. Such kinds of studies can be supported
by the use of primary cell cultures from various organisms and tissues, but we can also
take advantage of transformed cell lines derived from viral infections or even different
types of tumor. Overexpression of gene products can be an alternative to defining gene
function, to interfere with and search for a particular signal transduction pathway, or even
to titrate post-translational modifications of histones and/or endogenous peptides. At the
present time, gene transfer methodologies for gene expression are more reliable as they
are based on better knowledge of the parameters involved, even though some problems
remain unsolved. Study of gene regulation is probably the most widely used method to
understand the activity of eukaryotic regulatory elements. The list of such elements is still
growing, including classic examples such as promoters, enhancers, locus control regions
(LCRs) and, more recently, insulators [2, 4-7]. All of these studies are based on two main
components: (i) the use of measurable reporter genes and (ii) subsequent transfer to cells in
a transient or stable way. Plasmids carrying different reporter genes, like chloramphenicol
acetyl-transferase (CAT), and more recently luciferase (LUC) genes,
β
-galactosidase and
the green fluorescence proteins (GFPs), are all commercially available, which are efficient
and save time in studying the activity of control elements [4].
It is very important to mention that transient transfection experiments, even though they
are very instructive, may give inconsistent results, particularly when compared with the
same reporter vectors now integrated into the genome of the host cells. In other words, the
chromatin environment of an integrated reporter plasmid can give totally different results
compared with episomal vectors. In our experience, transient assays reproducibly determined
a silencer activity associated with non-coding sequences located at the 3
-end of the chicken
α
-globin domain. When the same sequences were tested in an integrated context, the silencer
contributed positively to the adjacent enhancer elements [8]. This clearly illustrates two
opposing activities, depending on whether the regulatory elements under study are located
on a chromatin context or not.
Thus, it is critical to understand, without any doubt, the different gene expression pro-
files within a cell. However, this is not a simple task because of the different networks and
redundancies used in nature to reach a highly regulated specific pattern of gene expression.
