Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
1999-11-12
2004-04-20
Mehta, Ashwin (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C435S320100, C435S468000, C800S278000
Reexamination Certificate
active
06723896
ABSTRACT:
BACKGROUND OF THE INVENTION
Transgenic techniques have become a powerful tool to address important biological problems in multicellular organisms, and this is particularly true in the plant field. Many approaches that were impossible to implement by traditional genetics can now be realized by transgenic techniques, including the introduction of homologous or heterologous genes into plants, with modified functions and altered expression patterns. The success of such techniques often depends upon the use of markers to identify the transgenic plants and promoters to control the expression of the transgenes.
Selectable markers are widely used in plant transformation. Historically such markers have often been dominant genes encoding either antibiotic or herbicide resistance (Yoder and Goldsbrough, 1994). Although such markers are highly useful, they do have some drawbacks. The antibiotics and herbicides used to select for the transformed cells generally have negative effects on proliferation and differentiation and may retard differentiation of adventitious shoots during the transformation process (Ebinuma et al., 1997). Also, some plant species are insensitive to or tolerant of these selective agents, and therefore, it is difficult to separate the transformed and untransformed cells or tissues (Ebinuma et al., 1997). Further, these genes are constitutively expressed, and there are environmental and health concerns over inserting such constitutively expressed genes into plants which are grown outside of a laboratory setting (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992).
A system to silence or remove such marker genes or other genes or to express them at only desired times would be very useful. Placing such genes under the control of an inducible or tissue-specific promoter has been accomplished. For example, transgenic plants expressing the ipt gene under the control of heat shock- (Medford et al., 1989), light- (Redig et al., 1996), copper- (McKenzie et al., 1998), tetracycline- (Redig et al., 1996; Faiss et al., 1997; Gatz et al., 1992) or dexamethasone- (Kunkel et al., 1999) inducible promoters have been used to study the biological effects of cytokinins. Other inducible systems include the heat-inducible expression system (Lyznik et al., 1995), the ethanol inducible system (Caddick et al., 1998), the ecdysone system (Martinez et al., 1999), and the TGV dexamethasone/tetracycline system (Bohner et al., 1999).
Excision of a marker gene by using the transposable element Ac has been performed, although this occurs at a very low frequency and after a long period of cultivation (Ebinuma et al., 1997). Another method for excising a gene is to use the Cre/lox system. The bacteriophage P1 Cre/lox site-specific recombination system (Dale and Ow, 1990; Odell et al., 1994) consists of two components: (i) a recombinase (CRE) and (ii) recombination sites (lox) at which the recombinase acts. The CRE gene encodes a 38 kDa recombinase which is able, without any other additional factors, to catalyze the recombination between two lox sites. A lox site consists of two inverted 13 bp repeats separated by an asymmetric 8 bp spacer where each inverted repeat acts as a binding site for CRE. The asymmetric nature of the 8 bp spacer gives a directionality to the lox site and determines the type of recombination event. The presence of two inverted lox sites leads to an inversion of the intervening DNA sequence whereas the presence of two directly repeated lox sites results in the excision of the intervening DNA sequence.
There are several site-specific recombination systems that have been shown to work in plants in addition to the described bacteriophage P1 Cre/lox system and these include: (i) the FLP-FRT system from
Saccharomyces cerevisiae
(O'Gorman et al., 1991), (ii) the GIN/gix system from bacteriophage Mu (Maeser and Kahmann, 1991) and (iii) the R/RS system from
Zygosaccharomyces rouxii
(Onouchi et al., 1991).
The FLP-FRT recombination system from
Saccharomyces cerevisiae
is based on site specific recombination by FLP recombinase on FLP recombination target sites (FRT). FRT consists of two inverted 13 base pair repeats and an 8 base pair spacer on which FLP recombinase acts. By inserting two directionally repeated FRT sites flanking a target gene it is possible, by addition of FLP recombinase, to excise the intervening DNA fragment by site-specific eviction. FLP recombinase mediated excision has also been shown to be reversible providing means for the introduction of DNA into specific sites in mammalian chromosomes (O'Gorman et al., 1991).
The Gin invertase encoded by bacteriophage Mu catalyzes the site-specific inversion of the G segment in the bacteriophage. The recombination sites (gix) are 34 base pairs in length and the two sites consist of two inversely oriented half-sites separated by two crossover regions. GIN acts on the gix sites by binding to the two half-sites and mediates DNA exchange and hence DNA inversion.
The R gene from pSR1 from
Zygosaccharomyces rouxii
encodes a recombinase that mediates site-specific recombination between two recombination sites (RS). The RS sites on pSR1 comprise a pair of inverted repeat sequences of 959 base pairs which contain the recombination sites (58 base pairs). Depending on the directionality of the RS sites, the R recombinase can catalyze, without any other additional factors, the excision (directionally repeated) or inversion (opposite orientation) of large DNA fragments (~200 kilobase pairs).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
SUMMARY OF THE INVENTION
The invention is directed to the use of an inducible promoter system in conjunction with a site-specific recombination system in order to (i) specifically activate transgenes at specific times and (ii) to excise and remove transgenes (e.g., antibiotic resistance markers) from trarsgenic plants once used and no longer needed. These “suicide” gene cassettes, including the recombination system itself, can therefore be evicted from the plant genome once their function has been exerted.
The system is based on the ability to temporally and spatially induce the expression of CRE recombinase which then binds to directly repeated lox sites flanking the transgene in question leading to the precise excision of the gene cassette. In order to test this system a construct was designed that allows in planta monitoring of precise excision events using the firefly luciferase (LUC) reporter gene as a marker for recombination.
REFERENCES:
patent: 5965791 (1999-10-01), Ebinuma et al.
patent: 6063985 (2000-05-01), Chua et al.
patent: 6326192 (2001-12-01), Sugita et al.
patent: 0 911 412 (1999-04-01), None
patent: WO 97 37012 (1997-10-01), None
Zuo et al., Nature Biotech., 2001, vol. 19, pp. 175-161.*
Sektas, Marian et al., “Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes.”Molecular Biotechnology, 9:1, pp. 17-24, 1998.
Albert, H. et al. “Site-specific integration of DNA into wild-type and mutant Iox sites placed in the plant genome”,The Plant Journal, 1995; 7(4):649-659.
Aoyama, T. et al. “A glucocorticoid-mediated transcriptional induction system in transgenic plants”,The Plant Journal, 1997; 11(3):605-612.
Aoyama, T. “Glucocorticoid-inducible Gene Expression in Plants”,Inducible Gene Expression in Plants(Ed. P. Reynolds), CAB International (Wallingford) (1999), pp. 43-59.
Baron, U. et al. “Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes”,Proc. Natl. Acad. Sci. USA, Feb. 1999; 96:1013-1018.
Böhner, S. et al. “Transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression”,The Plant Journal, 1999; 19(1):87-95.
Busch, M.A. et al. “Activation of a Floral Homeotic Gene in Arabidopsis”,Science, Jul. 23, 1999
Chua Nam-Hai
Moller Simon Geir
Zuo Jianru
Mehta Ashwin
Rothwell Figg Ernst & Manbeck P.C.
The Rockefeller University
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