Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-04-28
2003-06-24
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S288000, C800S292000, C800S293000, C800S294000, C800S306000, C800S317300, C435S069800, C435S320100, C435S419000, C435S468000, C435S469000, C435S470000
Reexamination Certificate
active
06583336
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel process for the production of transgenic organisms or transgenic cells, to transgenic organisms or transgenic cells obtainable by the process of the present invention, to the use of vectors comprising DNA encoding a recombination promoting enzymes for curing impairments caused by environmental influences in plants or plant cells and for gene therapy in mammals or mammalian cells, and to novel vectors.
The process of homologous recombination requires search for homology, recognition of sequence similarity, and strand exchange between two DNA molecules. In bacteria, these different steps are mediated by a single protein, the RecA protein (for review see:Roca and Cox, 1990), which plays a central role in the recombination pathway of
E. coli
. However, additional proteins are needed to initiate recombination and to resolve the intermediates created by RecA. Recombination is initiated by the generation of single-stranded DNA (ssDNA) and DNA ends in
E. coli
and presumably in all organisms. In
E. coli
, the combined action of the products of the recB, recC, and recD genes initiates a major recombination pathway (for review see: Dunderdale and West, 1994). ssDNA is recognised by RecA protein and double-stranded DNA (dsDNA) is actively searched for. Exchange of complementary strands leads to the formation of recombination intermediates (Holliday structures). The intermediates can be resolved by different pathways; the major one involves the action of the RuvA, RuvB, and RuvC proteins. All of the recombination proteins have to work in concert to complete recombination successfully. Proteins remarkably similar to RecA have been found in a number of eukaryotic cells such as budding yeast, fission yeast, humans, mice, chicken, and plants (Terasawa et al., 1995; for review see: Kowalczykowski and Eggleston, 1994). The best characterised ones are the Dmc1 and Rad51 proteins from Saccharomyces cerevisiae. In both cases the corresponding genes are essential for recombination and the proteins show considerable sequence homology to RecA. A comparison of the primary sequences of Dmc1 and several bacterial RecA proteins suggests that these proteins evolved from a single progenitor before the separation of prokaryotes and eukaryotes. In addition, Rad51 was shown to be structurally very similar to RecA. Rad51 forms DNA/protein filaments, strikingly similar in tertiary structure to those formed with RecA (Ogawa et al., 1993). While previous studies failed to show ATP-dependent homologous pairing and strand-exchange mediated by Rad51 (Shinohara, et al., 1992; Ogawa et al., 1993), more recent experiments have demonstrated these activities (Sung, 1994). Rad51 interacts with other proteins, e.g. Rad52 and Dmc1, so Rad51 may be part of a complex involved in recombination.
However, the complexity of these proteins strongly argues against their being simply a homologue as equivalent to the
E.coli
RecA protein. Accordingly, different modes of biological activity may be expected.
Various reports have been published focusing on the activity of
E. coli
RecA protein in animal and in particular in mammalian cells. Thus, Kido et. al, 1992 report on the introduction of functional bacterial RecA protein which was fused to the nuclear location signal of SV40 large T-antigen into mammalian cells. However, no functional studies of the introduced protein were carried out. WO 93/22443 deals with the targeting of exogenous polynucleotide sequences coated on
E. coli
RecA protein to chromosomal DNA of mammalian cells. This document shows that RecA protein coated oligonucleotides can efficently be targeted to correct chromosomal positions, RecA can stimulate extrachromosomal recombination, and RecA short DNA complexes can be used for gene targeting in mammalian cells. However, the authors failed to show stimulation of homologous recombination in living cells or an entire organism. Spivak et. al (1991) report the increased survival of HeLa cells upon treatment with RecA protein containing liposomes after irradiation. However, RecA stimulated survival was only marginal. Cerruti et al. report on the recombinatorial activity of
E.coli
RecA protein in plastids which, however, had no effect on DNA repair or cell survival, probably due to the fact that plastids have an own recombination promoting enzyme which is homologous to
E.coli
RecA. Thus, so far successful experiments with the goal of targeting RecA protein to eukaryotic nuclei which yield a significantly high recombinatorial activity to allow for the industrial applicability of such processes have not been carried out. For example, as regards plant cells, introduction of RecA/DNA complexes, in analogy to WO93/22443, in plant cells by PEG-mediated transformation turned out to be extremely difficult. These complexes exhibit an apparent toxicity and lead to cell death of nearly the total protoplast population. Also, Kido et. al, loc. cit., had reported on the failure to introduce RecA protein into the nuclei of mammalian cells. Therefore, in view of the prior art investigations it was highly questionable as to whether a recombination promoting enzyme such as RecA could be functionally introduced into the nuclei of eukaryotic cells and, furthermore, whether such an introduced RecA protein would indeed be able to enter the cell nucleus and actively promote recombination to an industrially applicable extent.
SUMMARY OF THE INVENTION
Thus, the technical problem underlying the present invention was to provide a process for the production of a transgenic organism or a transgenic cell, said process making use of a recombination promoting enzyme. The solution to said technical problem is provided by the embodiments characterised in the claims. Accordingly, the present invention relates to a process for the production of a transgenic organism or a transgenic cell comprising
(a) insertion
(aa) of a DNA into the genome of an organism or a cell, said DNA comprising a DNA which
(aaa) confers to the transgenic organism or the transgenic cell one or more desired characteristics; which
(aab) additionally encodes at least one selection marker expressible in said organism or said cell; and which
(aac) optionally encodes a recombination promoting enzyme or an enzymatically active derivative or part thereof, wherein the recombination promoting enzyme or the enzymatically active part thereof confers the or one of the desired characteristics; or, if (aac) does not apply,
(ab) of a recombination promoting enzyme or an enzymatically active derivative or part thereof in combination with said DNA (aa), into an organism or a cell;
(b) selection of transgenic organisms or cells, which have taken up said DNA or said DNA and said protein according to (a); and
(c) culturing of the desired transgenic organism or the desired transgenic cell in a suitable culture medium.
Thus, it is conceivable in accordance with the present invention that the recombination promoting enzyme confers the desired characteristic or is one of the desired characteristics. In the first instance, the method of the invention may yield, for example, plants with a hyperrecombinant phenotype which might be of use in plant breeding, plants more tolerant to environmental influences, for example, caused by UV or ozone, or plants more tolerant to DNA damage. In the second case, the recombination promoting enzyme may be used to introduce by promoting recombination a DNA sequence of interest into the genome of a cell or an organism. These transgenics are expected to improve the frequency of gene targeting and make this methodology applicable, for example, for plant breeding.
Further, the recombination promoting enzyme may be introduced into the cell or organism as encoded by a corresponding nucleotide sequence which, upon expression, yields said recombination promoting enzyme. Alternatively, the recombination promoting enzyme may be introduced into said cell or organism as such. In this case, the DNA to be inserted encodes a protein with the second or further desired
Klemm Manfred
Kosak Hans
Reiss Bernd
Schell Jeff
BASF Plant Science GmbH
Birch & Stewart Kolasch & Birch, LLP
Fox David T.
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