Biomedical Engineering Reference
In-Depth Information
mechanisms have, in particular, contributed to the understanding of such variability on gene
expression. This has led to designing of novel strategies for counteracting the silencing
effect of chromatin on random integrated genes, resulting in sustained and homogeneous
transgene expression.
7.1.4 Tissue-specific regulatory elements
For any successful gene transfer and expression analysis, the tissue- or cell-specificity should
be determined carefully. In addition, the strength of associated regulatory elements repre-
sents a complementary factor. Position effects of chromatin had been overcome by the use
of strong and dominant regulatory elements. Certain viral enhancer-promoter combina-
tions are largely sufficient to overcome position effects, like the cytomegalovirus (CMV)
promoter-enhancer elements (Rincon-Arano and Recillas-Targa, unpublished observations).
Unfortunately, these elements inherently possess a large spectrum of cell-type activity,
making transgene expression too non-specific. With the discovery of LCR in the human
β
-globin locus, particular excitement was generated in opening up the possibility of over-
coming position effects when those sequences were included in transgene vectors [13, 23].
The
β
-globin LCR provides strong erythroid-specific gene expression, and, in its absence,
these genes are subject to strong chromatin position effects. When linked to a reporter
gene, there is a copy-number-dependent gene expression in erythroid cells, independent
of the integration site [13, 23]. In other words, the presence of LCR had a positive and
dominant effect on transgene expression independent of the chromatin integration context
[7, 13, 24]. To date, a significant number of LCRs have been discovered, all of them
being tissue specific [25]. Based on their properties, the LCRs have been incorporated
into transgene designs in retroviral vectors and in the generation of transgenic mice [26,
27]. The principal restriction for the use of LCRs in recombinant gene expression is their
tissue-specificity.
7.1.5 Sustained expression and chromatin insulators
A more recent and attractive alternative for transgene expression is the use of insulator or
boundary elements [2, 6]. Initially discovered in
Drosophila
, insulators have emerged in
different chromatin domains. At the moment, the best-characterized insulator is the chicken
β
-globin insulator [2, 28]. Insulators are functionally defined based on two experimental
properties: (i) they are able to interfere with enhancer-promoter communication exclusively
when located between them and (ii) they have the capacity to protect a transgene, when
located on each side of the vector, against chromatin position effects, independent of the
genomic integration site [2, 6, 28]. Usually, insulators co-localize with constitutive DNase
I hypersensitive sites and, in general, they behave as neutral elements; that is, they are not
activators or repressors of transcriptional activity. All these features, and particularly the
ability to shield transgenes against chromatin position effects and progressive extinction of
expression, show the real potential of insulators in transgenesis and gene therapy. Thus,
the use of chromatin insulators, in combination with tissue-specific regulatory elements, is
getting closer to becoming a real means of protecting against position effects with direct
consequences in the expression patterns of transgenes and gene therapy vectors.
In conclusion, there are several methodologies that can be used for the study of gene
expression. Here, we first describe the method associated with transgene interchange,
