LLUÍS MONTOLIU'S LABORATORY at CNB



 

Research Interestsback

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.
 

in Spanish 

Research Summany of Dr. Lluis Montoliu's laboratory 2003-2004 (PDF format)

Last Update: 28 September 2005