Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1997-05-30
2004-08-10
Falk, Anne-Marie (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S013000, C800S014000, C800S018000
Reexamination Certificate
active
06774279
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the use of Flp recombinase to catalyze FRT site-specific DNA recombination in a transgenic non-human mammal, preferably a mouse.
2. Description of the Related Art
Site-specific recombinases are being developed as tools for genetic engineering because of their simplicity and precise activity in a variety of organisms. Two well studied recombinases are Flp and Cre. For use in vivo, a recombinase should be active in a transgenic non-human mammal. While Cre-mediated recombination has been successfully employed in trangenic mice, the utility of Flp recombinase in transgenic mice has not previously been established.
U.S. Pat. No. 4,959,317 discloses the use of the Cre-lox recombinase system in yeast and cultured mammalian cells, but not in transgenic mice. Other site-specific recombinases were not discussed.
U.S. Pat. No. 5,527,695 demonstrates the use of Flp recombinase in plants, but not in cultured mammalian cells or transgenic mice. A number of different site-specific recombinase systems are discussed; however, no guidance appears to be given for selecting among the different systems and their use in a transgenic mouse is not discussed.
Kilby et al. (1993) reviewed the demonstrated activities of different site-specific recombinases in cells and organisms. Table 1 shows that, to their knowledge, Flp recombinase activity in transgenic mice had not been accomplished.
Flp-mediated deletion was demonstrated in embryonic stem (ES) cells by Jung et al. (1993). Gu et al. (1993) compared the activity of Cre and Flp recombinases in ES cells and found “a major fraction of ES cells transiently transfected by the cre vector undergo Cre-loxP-mediated gene deletion (which is not the case in our hands if the related FLP/FRT system from yeast is used [Jung et al., 1993, and unpublished data])”. Both papers were contributed by the Rajewsky group, and the same group has exclusively used the Cre-loxP system in transgenic mice to inactivate endogenous genes, instead of Flp recombinase (Gu et al., 1994; Kühn et al., 1995). Thus, prior to the present invention, it was thought that using Cre recombinase was preferred over using Flp recombinase.
In view of the above teachings of the related art, it is an unexpected finding of the present invention that Flp recombinase can function in a developing mammal to catalyze FRT site-specific recombination.
Moreover, although Cre recombinase has been successfully used to create specific deletions in the mouse genome, the general utility of Cre to catalyze recombination is currently being established. Therefore, an additional method is needed for generating site-specific genetic alterations in the following ways: (1) a site-specific recombinase demonstrating a different dose-sensitivity could be used in situations where proper regulation of the recombination event cannot be achieved using Cre and (2) two site-specific recombinases could be used in vivo to engineer simultaneous or sequential recombination reactions (e.g., independent gene activation or inactivation events).
For example, site-specific recombinases may be used to activate expression of a tracer molecule to mark cell lineages. Factors that influence the determination of these cell lineages can be identified by analyzing these marked cells in the genetic background of various mutations, including mutations generated using the second recombinase system. Additionally, having access to two recombinase systems allows for efficient use of the first recombinase to generate a mutation, and the second recombinase to remove any selectable markers used in generating that mutation which, if left in place, would confound interpretation of the study. A second recombinase system is desired which exploits the ability of Flp recombinase to catalyze FRT-specific recombination in a transgenic non-human mammal which can be used alone or to expand the uses of the CrelloxP system.
The present invention provides a transgenic non-human mammal with sufficient Flp recombinase activity to catalyze recombination between FRT sequences, a transgenic non-human mammal containing FRT target nucleic acid which serves as an efficient substrate for Flp, a process of in vivo gene manipulation using the transgenic non-human mammals, and a genetic system comprised of the Flp transgenic non-human mammal and the FRT target transgenic non-human mammal which contains at least one FRT sequence.
BRIEF SUMMARY OF THE INVENTION
An object of the invention is to provide a transgenic non-human mammal with Flp recombinase activity useful for manipulation of the genome in the intact mammal.
Yet another object of the invention is to provide a process of in vivo genetic engineering using Flp recombinase activity to catalyze FRT site-specific recombination in a non-human mammal.
A further object of the invention is to provide a genetic system of the transgenic non-human mammal with Flp recombinase activity and at least one nucleic acid which is a substrate for Flp recombinase (e.g., the nucleic acid contains a FRT sequence). The nucleic acid may also contain a transgene for insertion into the genome of a non-human mammal, or a region which directs homologous recombination into the genome of a non-human mammal.
In one embodiment of the invention, a transgenic non-human mammal is provided which contains a Flp transgene integrated in its genome. Optionally, at least one Flp-recognition sequence is also integrated in the genome of the transgenic non-human mammal. The Flp-recognition sequence comprises FRT or a derivative thereof such as, for example, SEQ ID NO:14 or SEQ ID NO:15. A transgenic non-human mammal of the invention contains sufficient Flp recombinase activity in a cell to catalyze recombination between Flp-recognition sequences of the cell, chromosomal and/or extrachromosomal. Flp recombinase activity may be regulated by a chemical (e.g., exogenously administered drug, endogenous metabolite), the mammal's developmental stage, its body temperature, or tissue type of the cell.
The substrate for Flp recombinase activity is a Flp-recognition sequence. The genome of the transgenic non-human mammal may comprise one Flp-recognition sequence, two Flp-recognition sequences, or more than two Flp-recognition sequences. A chimeric or mosaic transgenic non-human mammal may contain cells with different numbers of Flp-recognition sequences due to Flp-mediated recombination; when a Flp-recognition sequence is integrated on only one of the pair of homologous chromosomes, the genome will be hemizygous for the Flp-recognition sequence.
Recombination between two Flp-recognition sequences integrated on different chromosomes results in translocation between those chromosomes. Such translocations are a common means of creating mutations that lead to developmental abnormalities or tumorigenesis.
Recombination between two Flp-recognition sequences in direct repeat orientation may cause excision of an intervening DNA sequence (e.g., a gene). Although such events are potentially reversible because Flp-mediated recombination is conservative, loss of the excised DNA sequence during cell division or by degradation makes the mutation irreversible. A null mutation in any gene may be created in this way, and the function of the gene studied in specific cells and/or at specific developmental stages.
Recombination between two Flp-recognition sequences in inverted repeat orientation may cause inversion of an intervening sequence or gene. As in Salmonella phase variation, inversion may cause activation or inactivation of a gene. If gene activity is detectable (e.g., selectable marker, histochemical marker, reporter gene), cell lineages may be traced by identifying recombination events that mark a cell and its descendants through detection of gene activation or inactivation. Cell lineages may be traced independent of gene activity, by monitoring differences in the integration site of the Flp substrate.
Recombination between a Flp-recognition sequence integrated on a chromosome and a F
Carnegie Institution of Washington
Falk Anne-Marie
LandOfFree
Use of FLP recombinase in mice does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Use of FLP recombinase in mice, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Use of FLP recombinase in mice will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3278667