LLUÍS MONTOLIU'S LABORATORY at CNB
Lluís Montoliu has been interested in transgenesis since 1989.
Transgenic animals represent one of most powerful tools to understand how
genes work during development. Further, they have shown to be unique in
modelling human diseases. However, a variable proportion of transgenic
animals usually do not display the appropriate pattern of expression. In
these cases, ectopic expression of the transgene can be detected and, what
is most frequent: its silencing. Thus, trying to understand how the expression
of a transgene could be improved and optimized is not only relevant for
basic biology but has tremendous importance in biotechnology and gene therapy
applications. Failure of a transgene to recapitulate the planned expression
programme has been correlated with its site of integration
within the host genome. There, a complex and not yet fully understood set
of events due to surrounding sequences (named position
effects) seem to be the cause of this undesirable transgene behaviour.
Position effects support the notion that genes are organized in chromosomes
as independent units referred to as expression
domains. These units often contain regulatory elements that might be
located far away from the body of the gene (promoter+exons+introns). However,
these distant elements have only been identified in very few cases (i.e.
the locus control region, LCR, of the human beta-globin locus) and are
usually not present in standard transgenic constructs. Current transgenes
are made in plasmids, bacteriophagues or cosmids and have an upper size
limit due to the capacity of heterologous DNA that these vectors can accommodate.
Thus, it was suggested that the transfer of bigger segments of genomic
DNA could help to establish the faithful expression pattern of the transgene.
Yeast artificial chromosomes (YACs) are vectors that can be used to
clone
and efficiently manipulate heterologous DNA sequences of megabase size.
In 1993, Prof. Schütz's laboratory and other groups pioneered the
transfer of YACs into the germline of mice. In Heidelberg, we could show
that transgenic mice made with a 250 kb YAC bearing the mouse tyrosinase
gene were able to completely rescue the albinism
of recipient animals with a position-independent and copy-number dependent
expression of the transgene. Those initial experiments and following served
to identify a Locus
Control Region (LCR) in the mouse tyrosinase gene. The further study
and characterization of this element will be the main research interest
of Lluís Montoliu's group. This will be not only important to understand
the regulation of the tyrosinase gene but can give valuable information
to the identification of expression domains and their application in biotechnology.
In addition, following a productive interaction with Dr. Glen Jeffery's
group at UCL in London, he has established a scientific collaboration to
study the role of tyrosinase (deficient in albinism) in eye and retina
development
by using transgenic animals.
FUNCTIONAL AND STRUCTURAL CHARACTERISATION OF GENOMIC BOUNDARIES
The recent availability of mammalian genomes (human and mouse) has shown
that the number of genes (~30.000) is likely to be lower than initially
anticipated. Opposite, the percentage of non-coding genomic regions (intergenic,
~95%), mostly dominated by different types of repetitive elements, is surprisingly
high. In addition, several types of regulatory elements are located within
these intergenic regions, which crucially cooperate in establishing and
identifying expression domains, defined as the set of neighbouring sequences
that restrict and guarantee the expression of a given gene in time and
space. Among those regulatory elements boundaries
stand out, in order to protect expression domains from regulatory elements
belonging to adjacent domains that could interfere with proper regulation
of gene expression. The structural and functional knowledge of these boundary
elements is not only interesting for basic biological processes but it
is fundamental to any biomedical or biotechnological gene transfer event
(animal models, gene therapy, production of recombinant proteins) in which
optimal heterologous expression is aimed at ectopic or extra-chromosomal
places of the genome. In our laboratory, we have two experimental models
specially suited to study gene transfer events: based on the tyrosinase
gene and the correction of albinism in transgenic mice, and the mammary
gland-specific whey-acidic protein gene, WAP. Previous studies have demonstrated
the existence of boundaries at the locus control region (LCR) of the tyrosinase
gene. Recent studies suggest the presence of analogous elements in the
WAP gene. In this project we will study in depth the structural and functional
properties of the tyrosinase boundary (and that of other possible boundaries
present in other places of this gene and other genes, such as WAP). Moreover,
we will systematically explore the potential use of these boundary elements
in different heterologous systems: mammalian cell cultures, retroviral
vectors, transgenic (mouse and Drosophila) animals, mammalian artificial
chromosomes, in order to evaluate whether the presence of boundaries associates
with more efficient gene transfer processes.
Research Summany of Dr. Lluis Montoliu's laboratory 2003-2004 (PDF format)
Last Update: 28 September 2005