Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Patent
1995-12-26
2000-03-21
Walsh, Stephen
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
435 691, 435 697, 435195, 4353201, 435325, 435 71, 435 18, 530350, 536 232, 536 234, 536 235, C07K 14435, C07K 14705, C12N 1512, C12N 1562
Patent
active
060404302
DESCRIPTION:
BRIEF SUMMARY
SPECIFICATION
The present invention relates to the technical field of genetic manipulation, and more specifically, to the use of recombination-mediated DNA rearrangements and to the use of the regulatory potential of nuclear receptors.
The use of site-specific recombinases (SSRs) to induce defined rearrangements of DNA has been described in a variety of organisms (1-12). These reports describe the introduction of a DNA construct that contains SSR target sites. Subsequent exposure to the SSR enzyme activity resulted in the DNA rearrangement determined by the disposition of the target sites (see reference 13 for a recent review of SSRs). Three SSRs have been used in this manner to date; FLP recombinase from the 2 .mu. episome of Saccharomyces cerevisiae (1,2,5,6,9,10), CRE recombinase from the Escherichia coli phage P1 (3,4,8,11,12) and R recombinase from pSR1 of Zygosaccharomyces rouxii (7). Amongst other SSR systems relevant to the invention described here are those listed in references 13 and 14, and SSRs from Kluyveromyces drosophilarium (15), Kluyveromyces waltii (16), .lambda. Int (17) and the Gin recombination system from phage Mu (18). The content of the above document is incorporated by reference.
For many applications in cells, organisms and cell-free in vitro systems, SSR induced DNA rearrangements must be regulated. Current implementation of the potential offered by SSRs is limited by the means available to regulate SSR activity. In experiments with cultured cells, unregulated SSRs have been used. For example, after introduction of SSR target sites into cells, recombination has been induced by subsequent introduction of either FLP recombinase by transfection of DNA (4,9) or injection of CRE recombinase protein (12). That is, the intended recombination event was regulated merely by the time of introduction of an appropriate macromolecule. Amongst other limitations, this precludes the creation and proliferation of homogeneous populations of cells that contain both the unrearranged target sites and the SSR and in which the recombination event can be induced after cell numbers have been expanded.
In experiments with transgenic animals, the issue of SSR regulation has been addressed by regulating the expression of an introduced SSR gene using the inducible heat-shock promoter in Drosophila (5) or a tissue-specific promoter in mice (11). Both approaches have limited applicability. Namely, heat-shock regulation of transgene expression is currently only useful in flies and no suitable counterpart is available for use in cell lines or vertebrates. Also, the use of a tissue-specific promoter to regulate transgene expression relies on the limited availability of suitable promoters and enhancers and the expression pattern achieved is confined to the times and places at which these tissue specific elements are active.
The problem underlying the present invention was to provide a regulated recombination system, wherein the disadvantages of the prior art are at least partially eliminated. More specifically, the problem was to provide a recombination system, wherein the recombination event can be induced independently from any tissue specific restrictions.
This patent application describes an invention that regulates SSR activity, rather than its expression. Thus any means of achieving and directing expression can be used, such as using ubiquitous or broadly active promoters and enhancers, as well as tissue specific or inducible promoters and enhancers.
One aspect of the present invention relates to a fusion protein, comprising a recombinase protein or a component of a recombinase complex, fused to part or all of a nuclear receptor, so that the amino acids that bind the ligand of said nuclear receptor are included, such that in cells or appropriate cell-free systems: (a) recombinase activity is inhibited in the absence of ligand binding to said ligand binding domain and (b) recombinase activity is induced or altered by binding of ligand to said ligand binding domain.
Preferably the recombinase activity is alter
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European Molecular Biology Laboratory (EMBL)
Pak Michael
Walsh Stephen
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