| Human lysyl hydroxylases: Characterization of a novel isoenzyme and its gene, determination of the domain structure of the lysyl hydroxylase polypeptides and generation of knock-out mice for the novel isoenzyme | ||
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Since the development of the technology needed to manipulate the germlines of animals almost two decades ago, a large number of transgenic animals have been produced worldwide for use in both basic and applied research. Animal models have now become valuable for studying human genetic diseases and for starting the testing of new therapeutic strategies, including gene therapy.
Of all the mammals studied by the early geneticists, the mouse became the first choice because of its small size, resistance to infection, large litter size and relatively rapid breeding time. The mouse is also genetically very similar to the human being, having a comparable genome size and number of genes and a similar basic physiology and patterns of development. A great number of inbred and genetically defined mouse strains are available nowadays.
Gene targeting, defined as the introduction of site-specific modifications into the genome by homologous recombination, has revolutionized the field of mouse genetics and allowed the analysis of diverse aspects of gene function in vivo. It is now possible to produce mice with specific genetic alterations ranging from simple gene disruptions and point mutations to chromosomal rearrangements, and even tissue-specific inducible gene targeting has become possible more recently (Müller 1999).
The development of embryonic stem cell (ES cell) technology (Evans & Kaufman 1981, Martin 1981), and the observation that introduced DNA can combine with its homologous chromosomal counterpart in ES cells (Thomas & Capecchi 1987), made it possible to apply exact modifications to the gene of interest. ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocyst-stage mouse embryos. Using standard gene technology, a targeting construct containing the desired alteration in the gene of interest and markers for selection flanked by sequences homologous to the endogenous target gene can easily be produced in vitro. The ES cells are then tranfected with the targeting construct, which will homologously recombine with the resident gene and introduce the mutation at the desired site in the genome (Matise et al. 2000).
In most cases, introducing a positive selection marker, which will disrupt the gene structure, is sufficient to achieve targeted inactivation of the gene. The most commonly used positive selection marker is a cassette carrying the neomycin resistance gene under the control of a strong promoter (von Melchner et al. 1992). In addition, a reporter gene can be introduced into the gene of interest in order to analyze the expression of the targeting construct in different tissues. The β -galactosidase gene is a widely used reporter gene which makes it possible to visualize the expression of specific gene products by X-gal staining on whole embryos and sections already in a heterozygous state (Hasty et al. 2000).