LLUÍS MONTOLIU'S LABORATORY at CNB
The experimentation with transgenic animals has shown temporal and spatial variability in transgene expression mainly due to position effects, related to the insertion of the transgene in the host genome. In this regard, the study of mechanisms regulating faithful transgene expression is essential for the development of animal models by genetic manipulation and, at the same time, allows increasing our knowledge on the organisation and structure of eukaryotic expression domains.
In our laboratory, we use two experimental models to analise regulatory
elements from genomic expression domains: the tyrosinase gene, specific
of melanocytes and of retinal pigment epithelium cells, and the whey acidic
protein (WAP) gene, mammary gland-specific during the lactation period
(in collaboration, the latter, with Dr. Eve Devinoy’s laboratory at INRA
in Jouy-en-josas, France, and Prof. M. Müller’s laboratory at the
Veterinary University of Vienna, Austria). We have used several natural
mutants of the tyrosinase gene and made a number of transgenic animals
with genomic fragment from both domains in order to uncover the most important
regulatory elements, which restrict tissue-specific expression and prevent
their ectopic activation. Among those identified elements, that are being
characterised both at the structural and functional level, there is a locus
control region (LCR), in the tyrosinase gene, and boundaries/insulators,
in both experimental systems. The principal task of boundaries is to delimit
the operative region of transcriptional regulators belonging to the domain
and to prevent interferences from activators and repressors of neighbouring
domains. Their existence can be uncovered with experiments in transgenic
animals in which we evaluate the capacity of these sequences to protect
a given heterologous transgene from position effects. In collaboration
with Dr. Ana Busturia’s laboratory (CBMSO, Madrid) we have generated and
analised multiples Drosophila transgenic lines carrying different construct,
with or without boundaries.
We are using also the tyrosinase experimental model to investigate the function of this protein, encoding the key enzyme in the melanin biosynthetic pathway, in mammalian retina development. Oculocutaneous albinism type I is caused by mutations in the tyrosinase gene. This genetic defect, affecting 1:10000 Europeans, is characterised by hypopigmentation and multiple abnormalities in the retina and the visual system. All these abnormalities disappear in transgenic mice obtained with tyrosinase functional constructs. In our laboratory, in collaboration with Dr. Glen Jeffery’s laboratory (UCL, London, UK) we study how, directly or indirectly, tyrosinase influences the normal development of mammalian retina, using several transgenic mouse models.
The study of the mechanisms, both functional and structural, implicated in obtaining an adequate pattern of transgene expression goes beyond the tyrosinase model being of capital relevance in projects of animal biotechnology and gene therapy, in which optimal expression must be guaranteed. In this sense, our laboratory has developed several transgenic constructs based in artificial chromosomes (BACs and YACs) aiming to improve heterologous expression in transgenic animals. In collaboration with Prof. F. Valdivieso’s laboratory (CBMSO, Madrid) we are producing a new animal model for the Alzheimer’s disease using a transgenic mouse prepared with a new artificial chromosome containing the APP gene.
Finally, in collaboration with a national pharmaceutical company, we
have generated and are analysing the phenotype of a mouse knock-out model
carrying a mutation, generated by homologous recombination in ES cells,
in the Sigma receptor Type I, involved in analgesia, schizophrenia and
psychosis phenomena.
Research Summany of Dr. Lluis Montoliu's
laboratory 2001-2002 (~1.2 Mb, PDF format)
