Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-09-22
2004-08-24
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S455000, C435S468000, C435S320100, C536S023100, C536S024300
Reexamination Certificate
active
06780644
ABSTRACT:
The present invention relates to the use of prokaryotic recombinase in eukaryotic cells, especially for transgenic work in eukaryotic cells.
It also relates to the use of prokaryotic beta recombinase for intramolecular recombination in eukaryotic cells and for mediating exclusively intramolecular reactions.
The use of the gene coding for beta recombinase for catalyzing site-specific resolution (deletion) of DNA sequences when the two target sequences (DNA sequences containing six sites) in a plasmid (extrachromosomal target) introduced in the cell by transfection or when the target sequences are integrated in the genome as chromatin associated structures, at several locations is also disclosed.
INTRODUCTION
Several methods have been developed allowing the manipulation of mammalian genomes in order to elucidate the relevance and function of particular genes of interest. Among them, the development of transgenic mouse strains and gene targeting technologies have turned out to be particularly useful (1, 2). These techniques have undergone a new advance with the characterization and application of site-specific recombinases (3).
Site-specific recombinases can be clustered into two major families. The first one (the Int family) comprises those enzymes that catalyze recombination between sites located either in the same DNA molecule (intramolecular recombination leading to resolution or inversion) or in separate DNA molecules (intermolecular recombination leading to integration) (4-7). The later property has been exploited to allow targeted insertion of specific sequences in precise locations (8, 9). The recombinases used for manipulating mammalian genomes are mainly the Cre and the Flp proteins, which belong to the Int family (3). The target sequences for these enzymes, named loxP sites for the Cre enzyme and FRT for Flp, consist on a short inverted repeat to which the protein binds. The recombination process is operative through long distances (up to 70 kb) in the genome. Using these enzymes, several authors have reported site- and tissue-specific DNA recombination in murine models (10-13), chromosomal translocations in plants and animals (14-16) and targeted induction of specific genes (17). For instance, expression of Cre from the lck proximal promoter leads to specific recombination in thymus (10). The gene encoding DNA polymerase beta has been tissue-specifically deleted using the same strategy (11). In a different approach, the SV40 large tumor antigen has been specifically activated in the lenses of mice, resulting in tumours at that location and not in the rest of the animal (18). The Cre-loxP strategy has also been used in combination with inducible promoters, like the case of the interferon gamma inducible promoter, that was used to provoke gene ablation in liver with high efficiency and with less extent in other tissues (12). This powerful site-specific recombination system, however, only allow the induction of a reduced number of recombination event in the same genome. Since each recombination reaction leaves a target sequence for the recombinase in the genome at the crossover site, and because integrases (e.g. Cre and Flp) can catalyze intermolecular recombination, the whole process may lead to undesired chromosomal rearrangements. This general limitation has been proposed to be partially solved by the use of mutant versions of the loxP sequences (18).
The second family of recombinases includes those enzymes that catalyse recombination only when the sites are located in the same DNA molecule (resolution and/or inversion); they are collectively termed resolvases/invertases (19). These site-specific recombinases, which are constrained to intramolecular reactions, could have an advantage over the Int family of recombinases. Beta recombinase, which belongs to the resolvases/invertases family, catalyses exclusively intramolecular deletions and inversions of DNA sequences located between two target sites for the recombinase, termed six (20-22). Each six site comprises 90 bp and is composed by two binding sites for the recombinase, termed I and II (20, 21, see FIG.
1
). Beta recombinase is encoded by the beta gene of the Gram-positive-broad-host-range plasmid pSM19035 (22-25).
The earlier known Cre system appears to be limited in the sense that it allows the induction of a reduced number of recombination events within the same mouse genome. When a conditional knockout mouse is made using this system, a loxP site remains in the genome and constitutes a potential target for another recombination event of unknown consequences. This problem would be fully overcome using an exclusively intramolecular site-specific recombinase.
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WO 0 92 19358, Nov. 12, 1992, International Application Published by WIPO; PCT Application.
Alonso Carlos Martinez
Alonso Juan C.
Bernad Antonio
Días Vincente
Rojo Fernando
Crouch Deborah
Dinsmore & Shohl LLP
Pharmacia Spain S.A.
Woitach Joseph
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