A novel method for the integration of foreign dna into eukaryoticgenomes

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DESCRIPTION

Novel method for the integration of foreign DNA into eukaryotic genomes.

The invention relates to the genetic modification of chromosomes. In particular, methods and compositions are provided for the integration of DNA into a eukaryotic genome.

Background of the invention

Various approaches have been used to integrate a DNA of interest into the genome of a plant. In the simplest method, DNA is introduced into a cell and randomly integrated into the genome by illegitimate recombination. A disadvantage of this method is that the positional effects due to random integration make gene expression difficult to analyze.

As an alternative to illegitimate recombination, integration must be directed to a particular site in the genome through the use of homologous recombination or site-specific recombination. In plants, where homologous recombination technology has not yet been developed, site-specific recombination is used to integrate a sequence of interest into an integration site that has previously been inserted into the host genome of the plant. If site-specific integration occurs by a single crossover event between a chromosome and a circular extrachromosomal replicon, the entire replicon will insert into the chromosome. When insertion of the entire replicon is not desired, a fragment of the replicon comprising the DNA of interest, flanked by target sites for a site-specific recombinase, can be introduced by a double reciprocal crossover event, into a chromosome having a single site. of integration corresponding to the target sites flanking the DNA of interest. In either case, integration is not efficient because it is reversible, that is, the integrated DNA can be cleaved by subsequent site-specific recombination between the target sites flanking the integrated DNA.

Various approaches have been taken to avoid cleavage of an integrated DNA. In one approach, the expression of a site-specific recombinase, such as Cre or FLP, is temporarily regulated. See, O'Gorman et al. (1991) Science 251: 1351-1355; Logie and Stewart (1995) Proc Nati Acad Sci 92: 5940-5944; Zhang et al. (1996) Nuc Acid Res 24: 543-548; Nichols et al. (1997) MolEndocinol 11: 950-961; and Feil et al. (1997) Biochem BiophyRes Comm 237: 752757. In these methods, the recombinase is expressed briefly, either transiently or inducible, in order to allow integration. However, cleavage of the integrated DNA can take place before the active recombinase disappears from the cell. Furthermore, intramolecular cleavage is kinetically favored over bimolecular integration. Therefore, integrated DNA is inherently unstable in the presence of recombinase.

A second approach reduces cleavage of the integrated DNA using pairs of individually mutated target sites on both the chromosome and the flanking DNA of interest. See Albert et al. (1995) Plant J 7: 649-659; Schlakey Bode (1994) Biochemistry 33: 12746-12751; O'Gorman et al. (1997) ProcNatl Acad Sci 94: 14602-14607; and Araki et al. (1997) Nuc Acid Res 25: 868-872. Recombination between individually mutated target sites results in doubly mutated target sites flanking the inserted DNA within the chromosome. Doubly mutated target sites are not well recognized by recombinase. Thus, the inserted DNA is excised from the chromosome by a reverse reaction only at low levels. However, this system has the disadvantage that individually mutated target sites often do not act as efficient recombination substrates, and thus the frequency of integration is reduced. Furthermore, transformants are unstable because cleavage can still take place, albeit at reduced frequency.

Ow et al. (1995), Critical Reviews in Plant Sciences 14 (3), 239-261, describe several site-specific recombination systems that have been shown to work in higher eukaryotic cells. These two-component systems consist of a single polypeptide recombinase and a short recognition sequence of less than 35 bp.

Accordingly, it is an object of the present invention to provide efficient methods for site-specific integration of DNA into eukaryotic genomes that avoid subsequent cleavage reactions and other non-productive recombination reactions.

Summary of the invention

Compositions and methods are provided for introducing a DNA of interest into a genomic integration site. In particular, the methods and compositions involve the use of a combination of target sites from two distinct site-specific recombinases, such as Cre and FLP, and the expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities against multiple target sites, and the nucleotide sequences and expression cassettes that encode those recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with DNA of interest that is flanked by corresponding target sites. Expression of either the novel chimeric recombinase or two site-specific recombinases in the eukaryotic cell results in the integration of the DNA of interest within the genome. The compositions and methods of the invention make use in the construction of stably transformed eukaryotic cells, and in particular plant cells.

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The methods result in efficient targeted genomic integration of DNA by site-specific recombination.

According to the invention, there is thus provided a recombinant protein comprising a first site-specific recombinase fused in-frame with a second distinct site-specific recombinase.

The invention also provides:

- a recombinant protein comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 6, 9 and 11;

- a nucleic acid molecule comprising a nucleotide sequence encoding a first site-specific recombinase fused in frame with a second distinct site-specific recombinase;

- a nucleic acid molecule comprising SEQ ID NO: 2, 4, 5, 7 or 8.

- a eukaryotic cell that has the nucleic acid molecule of the invention stably incorporated into its genome;

- a plant that has stably integrated within a chromosome at least one integration site comprising a target site for a first site-specific recombinase and a target site for a second distinct site-specific recombinase, wherein said target sites they are contiguous.

- a plant that has a nucleic acid molecule of the invention stably integrated into its genome;

- a method for integrating a DNA of interest into the genome of a eukaryotic cell, comprising:

(a) introducing into said eukaryotic cell a transfer cassette comprising said DNA of interest, wherein said DNA of interest is flanked by a target site for a first site-specific recombinase and a target site for a second site-specific recombinase distinct site, and said eukaryotic cell genome comprises at least one integration site comprising target sites corresponding to said target sites flanking said DNA of interest; and, (b) providing in said eukaryotic cell a recombinant protein comprising said first recombinase, an active derivative of said first recombinase, or an active fragment of said first recombinase fused in frame with said second distinct recombinase, an active derivative of said second distinct recombinase, or an active fragment of said second distinct recombinase, wherein said first and said second recombinase, said active derivative, or said active fragment catalyzes a conservative site-specific recombination event, in which the DNA of interest integrates into the genome at the site of integration.

- a method for integrating a DNA of interest into the genome of a eukaryotic cell, comprising:

(a) introducing into said eukaryotic cell a transfer cassette comprising said DNA of interest, wherein said DNA of interest is flanked by a target site for a first site-specific recombinase and a target site for a second site-specific recombinase distinct site, and said eukaryotic cell genome comprises at least one integration site comprising said target sites corresponding to said target sites flanking said DNA of interest; and, (b) providing in said cell said first recombinase, an active derivative of said first recombinase, or an active fragment of said first recombinase; and, said second distinct recombinase, an active derivative of said second recombinase, or an active fragment of said second distinct recombinase, wherein said first and said second recombinases, said active derivative, or said active fragment catalyze a specific recombination event of conservative site; Y

- a transformed seed of a plant of the invention.

Brief description of the figures

Figure 1 schematically represents plant transformation vectors, PHP13164 and PHP13147, for the expression of moCRE recombinase and Cre: FLPm recombinase, respectively.

Figure 2 graphically depicts activation of GUS expression by FLPm or CRE: FLPm mediated cleavage of a sequence flanked by FRT sites separating a ubiquitin promoter and the GUS open reading frame.

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Figure 3 graphs the activation of GUS expression by CRE: FLPm-mediated cleavage of a sequence flanked by loxP sites that separate the ubiquitin promoter and the GUS open reading frame.

Detailed description of the invention

Compositions and methods are provided for site-specific integration of DNA into predetermined genomic integration sites in a host genome. The invention envisions the use of chimeric recombinases that catalyze site-specific recombination between target sites originating from different site-specific recombination systems. Such a dual-function chimeric recombinase ensures that the two foreign DNA ends do not ligate with each other, but instead recombine with their analogous pairing target sites that are housed in genomic DNA. The methods facilitate the directional targeting of nucleotide sequences and desired genes within corresponding integration sites previously introduced into the genome.

In the methods of the invention, a combination of target sites for two site-specific recombinases are introduced into the genome of an organism of interest, establishing an integration site for the insertion of nucleotide sequences of interest. For the purposes of the invention, an integration site will comprise flanking target sites in which the target sites correspond to the recombination sites for two distinct site-specific recombinases. These target or recombination sites may flank other nucleotide sequences or may be contiguous. Methods for the production of transgenic plants containing specific recombination sites integrated into the plant genome are described in co-pending provisional patent application, serial number 60 / 065,627, entitled "Compositions and Methods for Modification Plant Genetics ”, filed November 18, 1997, and which is incorporated herein by reference. Once a stable plant or cultured tissue is established, a transfer cassette comprising a DNA of interest, flanked by target sites corresponding to those of the genome integration site, is introduced into the stably transformed plant or tissues. in the presence of a chimeric recombinase with specificities for each of the target sites. Alternatively, two different recombinases corresponding to the target sites may be present in the cell in place of one chimeric recombinase. This procedure results in an exchange of nucleotide sequences between the two identical target sites of the genomic integration site and the transfer cassette.

Thus, the invention provides a method for integrating a DNA of interest within the genome of a eukaryotic cell, comprising:

(a) transforming said cell with a transfer cassette comprising said DNA, wherein said DNA is flanked by a target site for a first site-specific recombinase and a target site for a second site-specific recombinase, and said genome contains an integration site comprising target sites corresponding to said target sites flanking said DNA; and (b) providing in said cell a recombinant protein comprising said first recombinase fused in frame with said second recombinase.

The invention further provides a method for integrating a DNA of interest into the genome of a eukaryotic cell, comprising:

(a) transforming said cell with a transfer cassette comprising said DNA, wherein said DNA is flanked by a target site for a first site-specific recombinase and a target site for a second site-specific recombinase, and said genome contains an integration site comprising target sites corresponding to said target sites flanking said DNA; and (b) providing in said cell said first recombinase and said second recombinase.

By "site-specific recombinase" is meant any enzyme that catalyzes conservative site-specific recombination between its corresponding recombination sites. For reviews of site-specific recombinase, see Sauer (1994) Current Opinion in Biotechnology 5: 521-527; and Sadowski (1993) FASEB 7: 760-767.

The first and second site-specific recombinases can be full-length recombinases and / or active fragments or derivatives thereof. Site-specific recombinases useful for creating the chimeric recombinases of the invention include recombinases of the integrase family, derivatives thereof, and any other naturally occurring or recombinantly produced enzyme or derivatives thereof, that catalyze conservative site-specific recombination between specified DNA sites. The integrase family of recombinases has more than thirty members and includes FLP, Cre, Int, and R. Preferably, the recombinases do not need cofactors or a supercoiled substrate. Most preferably, the recombinases are Cre and FLP. The site-specific recombination systems of the bacteriophage P1, loxP-Cre, and the Saccharomyces 2µ FRT / FLP plasmid have been extensively studied and their uses are well known to those of skill in the art. Cre and FLP are known to function in a variety of organisms, including bacteria, yeast, Drosophila, mammals, and monocot and dicot plants. Furthermore, these recombinases do not need auxiliary factors to function.

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The site-specific recombinases and the sequences that encode them that are used in the methods and compositions of the invention can be variants of naturally-occurring recombinases and the genes that encode them. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refer to nucleic acids that encode identically or conservatively modified variants of amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be modified to any of the corresponding codons described without modifying the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine) can be modified to give a functionally identical molecule.

As for amino acid sequences, one of skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that modifies, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" in which the modification results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be both modified. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 modifications can be made. Conservatively modified variants normally provide biological activity similar to that of the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzymatic activity, or ligand / receptor binding generally represent at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables that provide functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for each other:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); Y

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See Creighton (1984) Proteins, WH: Freeman and Company.

Rather than using full-length recombinases, functional fragments of site-specific recombinases can be used in the methods and compositions of the invention. Functional fragments of site-specific recombinases can be identified using a variety of techniques. For example, functional fragments of the FLP protein can be identified by their ability, upon introduction into cells containing appropriate FRT substrates, to catalyze site-specific recombination and result in cleavage of an assayable marker gene.

A general approach to such functional analysis involves subcloning DNA fragments from a genomic clone, cDNA clone, or synthesized gene sequence within an expression vector, introducing the expression vector into a heterologous host, and selecting for the recombination product. (i.e., using restriction analysis to verify the recombination product at the nucleic acid level, or relying on a recombination assay system as described above). Methods for generating fragments of a cDNA or genomic clone are well known. Variants of an isolated DNA encoding a site-specific recombinase can be produced by nucleotide deletion, addition, and / or substitution. Such variants can be obtained, for example, by oligonucleotide-directed mutagenesis, binding sequence selection mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See, for example, Ausubel, Current Protocols In Molecular Biology, Wiley Interscience (1990) pages 8.0.3-8.5.9, and McPherson (ed.), Directed Mutagenesis: A Practical Approach, (IRL Press, 1991).

The dual-function recombinant proteins of the invention comprise a first site-specific recombinase fused in frame with a second site-specific recombinase. It will be recognized that in the methods of the invention, the recombinases comprising the chimeric recombinase must correspond to the target sites of the transformed organism and the target cassette. That is, if FRT and loxP sites are used, a chimeric FLP: Cre recombinase will be needed.

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The open reading frames encoding the first and second recombinases can be fused directly to each other or can be joined by a joining sequence that maintains the correct reading frame of the chimeric recombinase. It is understood that recombinases can be fused at the amino terminus to the carboxyl terminus, at the amino terminus to the amino terminus, or at the carboxyl terminus to the amino terminus.

Genes encoding chimeric site-specific recombinases and recombination sites can be produced using conventional recombinant methods, synthetic techniques, or combinations thereof. The use of cloning vectors, expression vectors, adapters, and binding sequences is well known in the art and can be found in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^to ed. (Cold Spring Harbor, New York, 1989). A variety of strategies are available for ligation of DNA fragments, the choice of which depends on the nature of the terminations of the DNA fragments and the choice of which should be readily made by those skilled in the art. The yeast (Saccharomyces cerevisiae) FLP recombinase gene is commercially available in plasmid pOG44 from Stratagene Cloning Systems (11011 North Torrey Pines Road, La Jolla, CA 92037). For description of the FLP gene and various nucleic acids, see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog'97 (Arlington Heights, IL). Similarly, the sequences of many other site-specific recombinases and their analogous recombination sites are publicly or commercially available. Genes encoding FLP and Cre can also be obtained, for example by synthesis of genes with long oligonucleotides that prime each other. See, for example, Ausubel et al. (eds.), Current Protocols In Molecular Biology, pages 8.2.8 to 8.2.13, Wiley Interscience (1990). Also see Wosniak et al. (1987) Gene 60: 115. Furthermore, current techniques using the polymerase chain reaction provide the ability to synthesize genes as large as 1.8 kilobases in length (Adang et al. (1993) Plant Mol. Biol. 21: 1131; Bombat et al. (1993) PCR Methods and Applications 2: 266).

When nucleic acid is synthetically prepared or modified, advantage can be taken of the known codon preferences of the intended host in which the nucleic acid is to be expressed. For example, although the nucleic acid sequences of the present invention can be expressed in both monocot and dicot plant species, the sequences can be modified to account for specific codon preferences and monocot or dicot GC content preferences since these preferences have been shown to differ (Murray et al. (1989) Nucl. Acids Res. 17: 477-498; and Campbell et al. (1990) Plant Physiol. 92: 1). Thus, the corn preferred codon for a particular amino acid can be derived from known corn gene sequences. Maize codon usage for 28 maize plant genes is listed in Table 4 of Murray et al., Cited above.

Examples of genes encoding recombinases, using preferred corn codons include, FLPm, described in co-pending patent application 08 / 972,258; the contents of which are incorporated herein by reference, and moCre, shown in SEQ. ID NOS. 1 and 2. FLPm is derived from the Saccharomyces 2 µ plasmid FLP recombinase, but is encoded by a nucleic acid sequence using corn-preferred codons. Although the FLPm nucleic acid sequence includes codons preferred for amino acid expression in corn, it is understood that a useful sequence may contain codons that appear in corn less frequently than the highest reported corn codon frequencies. Examples of nucleic acids encoding chimeric recombinases include Cre: FLPm (SEQ. ID NO. 4), moCre: FLPm (SEQ. ID NO. 5), Cre: FLP (SEQ. ID NO. 7), and FLPm: Cre ( SEQ. ID NO. 8).

The invention also provides expression cassettes containing a nucleic acid sequence encoding a chimeric site-specific recombinase, operably linked to a promoter that drives expression in a eukaryotic cell. Preferably the promoter is a plant promoter. For example, the plant expression vector PHP13147, shown in Figure 1, contains an expression cassette for Cre: FLPm, in which the gene encoding chimeric recombinase is operably linked to a ubiquitin promoter. As used herein, "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence that corresponds to the second sequence. . Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous and, in which it is necessary to join two protein coding regions, contiguous and in the same reading frame.

As used herein "promoter" refers to a region of DNA 5 'from the start of transcription and is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter that can initiate transcription in plant cells. Exemplary plant promoters include, but are not limited to, those derived from plants, plant viruses, and bacterial genes that are expressed in plant cells such as those from Agrobacterium or Rhizobium. Both heterologous and non-heterologous (ie, endogenous) promoters can be employed to direct the expression of a sequence encoding a site-specific recombinase. The promoter can be constitutive, inducible, or tissue specific.

Many different constitutive promoters can be used in the present invention. Example constitutive promoters include promoters from plant viruses such as the CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812) and promoters from genes such as rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); histone H3 from maize (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285 and Atanassova et al. (1992) Plant Journal 2

ES 2 256 972 T3 (3): 291-300); the 1 '- or 2' promoter derived from Agrobacterium tumefaciens T-DNA, the Smas promoter, the cinnamic alcohol dehydrogenase promoter (US Patent No. 5,683,439), the Nos promoter, the Pemu promoter, the rubisco, the GRP1-8 promoter, and other transcription initiation regions of various plant genes known to those of skill. The ALS promoter, a 5-prime Xbal / Ncol fragment of the ALS3 structural gene from Brassica napus (or a nucleotide sequence having substantial sequence similarity to said Xbal / Ncol fragment), represents a particularly useful constitutive promoter. (See copending US patent application 08 / 409,297 to Pioneer Hi-Bred International and corresponding US patent number 5,659,026 issued August 19, 1997).

A variety of inducible promoters can be used in the present invention. See Ward et al. (1993) Plant Mol. Biol. 22: 361-366. Example inducible promoters include those of the copper-responsive ACE1 system (Mett et al. (1993) PNAS 90: 4567-4571); the maize In2 gene that responds to benzenesulfonamide herbicidal antidotes (Hershey et al. (1991) Mol. Gen. Genetics 227: 229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243: 32-38); the Adhl promoter which is inducible by hypoxia or cold shock, the Hsp70 promoter which is induced by heat shock, and the PPDK promoter which is inducible by light; or the Tn10 Tet repressor (Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237. A particularly preferred inducible promoter is the promoter that responds to an inducing agent that plants do not typically respond to. An example inducible promoter is the inducible promoter of a steroid hormone gene whose transcriptional activity is induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421).

Examples of promoters under developmental control include promoters that only initiate transcription, or preferably, in certain tissues, such as leaves, roots, fruit, seed, or flowers. The functioning of a promoter can also vary depending on its location in the genome. Thus, an inducible promoter can become fully or partially constitutive at certain locations.

The chimeric recombinase must be expressed in the plant cell in order to integrate the DNA of interest into the host chromosome. Consequently, the expression cassette encoding the site-specific recombinase can be delivered cis in the DNA of interest; in trans on a host chromosomal or extrachromosomal replicon; or it can be transferred to the host and transiently expressed near the time recombination is desired.

Compositions of the invention include transfer cassettes comprising nucleotide sequences encoding the chimeric recombinases of the invention. By "transfer cassette" is meant any nucleotide sequence that can be used to transform a cell of interest. For example, the transfer cassette can be a separate replicon such as a plasmid, shuttle vector, Ti plasmid, viral vector, or the like. Alternatively, the transfer cassette could be a nucleic acid that cannot replicate independently, although it could be transferred into an organism of interest by a variety of transformation protocols, such as particle bombardment, electroporation, and the like. Thus, the invention provides a transfer cassette comprising a nucleotide sequence encoding a recombinant protein comprising a first site-specific recombinase fused in-frame with a second site-specific recombinase, wherein said nucleotide sequence is linked operatively to a promoter that drives expression in a eukaryotic cell.

In the compositions and methods of the invention, the DNA of interest is flanked by target sites for two distinct site-specific recombinases. By "flanked by" is meant that the recombination or target sites may be directly contiguous to the DNA of interest, or there may be one or more introns present between one or both ends of the DNA of interest and the site-specific recombination sites. Introns of particular interest would include junction sequences, adapters, selectable markers, and / or other sites that aid in vector construction or expression cassette and analysis for a gene of interest. Target sites for site-specific recombinases are known to those of skill in the art and are discussed in co-pending provisional patent application 60 / 065,613. Examples of target sites include, but are not limited to, FRT, FRT1, FRT5, FRT6, FRT7, other FRT mutants, loxP, loxP mutants, and the like. See, for example, Schlake and Bode (1994) Biochemistry 33: 12746-12751; Huang et al. (1991) Nucleic Acids Research 19: 443-448; Sadowski (1995) In Progress in Nucleic Acid Research and Molecular Biology vol.51, pages 53-91; Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society of Microbiology, Washington DC, pages 116-670; Dixon et al. (1995) 18: 449-458; Umlauf and Cox (1988) The EMBO Journal 7: 1845-1852; Buchholz et al. (1996) Nucleic Acids Research 24: 3118-3119; Kilby et al. (1993) Trends Genet. 9: 413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594; Albert et al. (1995) The Plant J. 7: 649-659: Bayley et al. (1992) Plant Mol. Biol. 18: 353-361; Odell et al. (1990) Mol. Gen. Genet. 223: 369-378; Dale and Ow (1991) Proc. Natl. Acad. Sci. USA 88: 10558-105620; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91: 1706-1710; Stuurman et al. (1996) Plant Mol. Biol. 32: 901-913; and Dale et al. (1990) Gene 91: 79-85.

By "target site for a site-specific recombinase" is meant a DNA sequence that is recognized by a particular site-specific recombinase. A variety of recombination sites are known to those skilled in the art and can be used in the methods and compositions of the invention. The site may have the sequence of the related site for a given recombinase, or it may be modified, as long as it can act as a recombination site. The site may contain the minimal sequences necessary for recombination, or it may contain additional sequences that promote recombination. Examples of recombination sites for use in the invention are known in the art and include FRT sites and loxP sites. (See, for example, Schlake and Bode (1994) Biochemistry

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33: 12746-12751; Huang et al. (1991) Nucleic Acids Research 19: 443-448; Paul D. Sadowski (1995) In Progress in Nucleic Acid Research and Molecular Biology vol. 51, pages 53-91; Michael M. Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society of Microbiology, Washington DC, pages 116-670; Dixon et al. (1995) 18: 449458; Umlauf and Cox (1988) The eMbO Journal 7: 1845-1852; Buchholz et al. (1996) Nucleic Acids Research 24: 31183119; Kilby et al. (1993) Trends Genet. 9: 413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594; Albert et al. (1995) The Plant J. 7: 649-659: Bayley et al. (1992) Plant Mol. Biol. 18: 353-361; Odell et al. (1990) Mol. Gen. Genet. 223: 369-378; Dale and Ow (1991) Proc. Natl. Acad, Sci. USA 88: 10558-105620; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91: 1706-1710; Stuurman et al. (1996) Plant Mol. Biol. 32: 901-913: Hardey et al. (1980) Nature 286: 860-864; Sauer (1994) Current Opinion in Biotechnology 5: 521-527; and Dale et al. (1990) Gene 91: 79-85.

Each loxP and FRT site contains two 13 base pair inverted repeats flanking an 8 base pair spacer. The FRT site contains an additional 13 base pair nonessential repeat. The sequences of the loxP and FRT sites are shown in SEQ. ID NO.1 and SEQ. ID NO. 2. A minimal FRT site (SEQ ID No. 10) comprising two repeats of 13 base pairs, separated by an 8 base spacer, is:

5'GAAGTTCCTATTC [TCTAGAAA] GTATAGGAACTTC3 'in which the nucleotides within the brackets indicate the spacer region. Nucleotides in the spacer region can be substituted with a combination of nucleotides, as long as the two 13-base repeats are eight nucleotides apart. FLP is a site-specific, conservative recombinase that can catalyze the inversion of a nucleic acid sequence placed between two inversely oriented FRTs; recombination between two molecules each containing a FRT site; and cleavage between FRT sites. The core region is not symmetric, and its asymmetry dictates the directionality of the reaction. Recombination between inverted FRT sites causes inversion of a DNA sequence between them, while recombination between directly oriented sites leads to DNA cleavage between them.

Nucleotide sequences containing a DNA of interest flanked by target sites, transfer cassettes for two different site-specific recombinases, and vectors carrying these sequences can be constructed using standard molecular biology techniques. See, for example, Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989).

Techniques for transforming a wide variety of eukaryotic cells, including higher plant species are well known and described in the technical, scientific and patent literature. See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). These methods are useful for transforming a plant cell with the chimeric recombinase expression cassettes of the invention and DNA of interest flanked by target sites for chimeric recombinase. The expression cassette encoding the site-specific recombinase may be present in the plant genome prior to transformation of the DNA of interest, or it may be transformed within the plant about the time of transformation with the T-DNA into the cell. vegetable so that it will express itself temporarily. For example, the DNA construct can be introduced directly into plant genomic DNA using techniques such as electroporation, PEG poration, particle bombardment, administration of silicone fibers, or microinjection of embryogenic plant cell protoplasts or calli.

Agrobacterium tumefaciens-mediated transformation techniques are well described in the scientific literature. See, for example, Horsch et al., Science 233: 496-498 (1984), Fraley et al., Proc. Natl. Acad. Sci. 80: 4803 (1983), Kado, (1991), Crit. Rev. Plant Sci. 10: 1 and Moloney et al. (1989), Plant Cell Reports 8: 238. Although Agrobacterium is useful mainly in dicots, certain monocots can be transformed by Agrobacterium. For example, Agrobacterium transformation of corn is described in US Patent No. 5,550,318. Other methods of agroinfection include Agrobacterium rhizogenes mediated transformation (see, for example, Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, CP, and Draper, J ,. In: DNA Cloning, Vol. II, DM Glover, Ed., Oxford, IRI Press, 1985), application PCT / US87 / 02512 (WO 88/02405 published April 7, 1988) describes the use of A.rhizogenes strain A4 and its plasmid Ri, together with the A. tumefaciens vectors, pARC8 or pARC16.

Vectors and optimized methods for Agrobacterium-mediated transformation of plants of the Graminae family, such as rice and corn are described in Heath et al. (1997) Mol. Plant-Microbe Interact. 10: 221-227; Hiei et al. (1994) Plant J. 6: 271-282 and Ishida et al. (1996) Nat. Biotech. 14: 745-750.

The transformation efficiency of maize is affected by a variety of factors including the types and stages of infected tissue, Agrobacterium concentration, tissue culture medium, Ti vectors, and maize genotype. Superbinary vectors carrying vir genes from Agrobacterium strains A281 and A348 are useful for high-efficiency transformation of monocots.

The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al., Embo J. 3: 2717-2722 (1984). Electroporation techniques are described in Fromm et al., Proc. Natl. Acad. Sci. 82: 5824 (1985). Ballistic transformation techniques are described in Klein et al., Nature 327: 70-73 (1987).

Viral means for introducing DNA into mammalian cells are known in the art. In particular, various vector systems are known for the introduction of foreign or native genes into mammalian cells.

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These include the SV40 virus (see, for example, Okayama et al. (1985) Molec. Cell Biol. 5: 1136-1142); bovine papillomavirus (see, for example, DiMaio et al. (1982) Proc. Natl. Acad. Sci. USA 79: 4030-4034); adenovirus (see, for example, Morin et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4626; Yifan et al. (1995) Proc. Natl. Acad. Sci. USA 92: 1401-1405; Yang et al. (1996) Gene Ther 3: 137-144; Tripathy et al. (1996) Nat. Med. 2: 545-550; Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2581-2584; Rosenfeld et al. (1991) Science 252: 431-434; Wagner (1992) Proc. Natl. Acad. Sci. USA 89: 6099-6103; Curiel et al. (1992) Human Gene Therapy 3: 147-154; Curiel (1991) Proc. Natl. Acad. Sci. USA 88: 8850-8854; LeGal LaSalle et al. (1993) Science 259: 590-599; Kass-Eisler et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1.1498-11502); adeno-associated viruses (see, for example, Muzyczka et al. (1994) J. Clin. Invest. 94: 1351; Xiao et al. (1996) J. Virol. 70: 8098-8108); herpes simplex virus (see, for example, Geller et al. (1988) Science 241: 1667; Huard et al. (1995) Gene Therapy 2: 385-392; US Patent No. 5,501,979); retrovirus-based vectors (see, for example, Curran et al. (1982) J. Virol. 44: 674-682; Gazit et al. (1986) J. Virol. 60: 19-28; Miller, AD (1992) Curr Top Microbiol Immunol 158: 1-24, Cavanaugh et al. (1994) Proc Natl Acad Sci USA 91: 7071-7075, Smith et al. (1990) Molecular and Cellular Biology 10: 3268-3271). See also, Wu et al. (1991) J. Biol. Chem. 266: 14338-14342; Wu and Wu J. Biol Chem. (1988) 263: 14621-14624; Wu et al. (1989) J. Biol. Chem. 264: 16985-16987; Zenke et al. (1990) Proc. Natl. Acad. Sci. USA 87: 3655-3659; Wagner et al. (1990) 87: 3410-3414.

DNA can also be introduced into plants by direct DNA transfer into pollen as described in Zhou et al., Methods in Enzymology, 101: 433 (1983); D. Hess, Intern Rev. Cytol., 107: 367 (1987); Luo et al., Plant Mol. Biol. Reporter, 6: 165 (1988). Expression of genes encoding polypeptide can be obtained by injection of DNA into reproductive organs of a plant as described in Pena et al., Nature, 325.:274 (1987). DNA can also be injected directly into cells of immature embryos and the dried embryos rehydrated as described in Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Erpo 1986, Butterworth, Stoneham. Mass., Pages 27-54 (1986). A variety of plant viruses that can be used as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, bromine mosaic virus, and tobacco mosaic virus.

Plant cells stably transformed with a chimeric recombinase expression cassette can be regenerated, for example, from single cells, callus tissue, or leaf discs according to plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully grown to generate a complete plant. Regeneration of plants from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillilan Publishing Company, New York, pages 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pages 21-73 (1985).

Regeneration of plants containing the recombinant genes can be accomplished as described in Horsch et al., Science, 227: 1229-1231 (1985). In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces shoot regeneration in the plant species being transformed as described in Fraley et al., Proc. Natl. Acad. Sci. USA, 80: 4803 (1983). This procedure typically produces shoots in two to four weeks and these transforming shoots are then transferred to an appropriate root inducing medium containing the selection agent and an antibiotic to prevent the growth of bacteria. The transgenic plants of the present invention can be fertile or sterile.

Regeneration can also be obtained from callus, explants and plant organs, or parts thereof. Such regeneration techniques are generally described in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants from either single plant protoplasts or diverse explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth procedure includes the steps of selecting transforming cells and shoots, rooting the transforming shoots and growing the seedlings in the soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3^to edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988).

One of skill will recognize that after DNA, such as a chimeric recombinase expression cassette or a target site for a chimeric recombinase, is stably incorporated into transgenic plants and confirmed to be functional, it can be introduced into other plants by sexual interbreeding. . Any of several conventional breeding techniques can be used, depending on the species to be crossed.

The methods and compositions of the invention are useful for integrating a DNA of interest into the genome of any host cell, including a plant host. As used herein, the term "plant" refers to whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, and plant cells, and progeny thereof. Plant cell as used herein includes, without limitation, suspension cultures of seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants arranged against transformation techniques, including both monocotyledonous and dicotyledonous plants. A particularly preferred monocot is corn. Other monocots of particular interest include wheat, rice, barley, sorghum, and rye. Dicots of particular interest include soybeans, brassica, sunflower, alfalfa, and safflower.

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Due to the use of chimeric site-specific recombinases and target sites provided herein, cells transformed by the methods of the invention can be distinguished from other transformation methods since the modified cells of the invention will contain nucleotide sequences of interest inserted within of the genome flanked by target sites for different recombinases.

The following examples are presented by way of illustration and not by way of limitation.

Experimental part Example 1

Construction of Vectors Containing a DNA of Interest Flanked by Target Sites for a Chimeric Site-Specific Recombinase

DNA fragments containing a DNA of interest flanked by loxP and FRT target sites are constructed by either synthesizing, pairing and ligating complementary oligonucleotides or by creating primers for PCR amplification of a DNA of interest containing loxP and FRT sites in addition to sites restriction tags useful for cloning into a vector of choice.

For example, long PCR primers can be designed in which the 3 'end of the primer hybridizes to the 5' end of DNA of interest and the 5 'end of the primer further contains useful loxP and FRT sites and cloning site. The resulting PCR product is digested with the appropriate restriction enzyme and inserted into an appropriate vector.

Example 2

Cleavage of the FRT site by FLPm and the chimeric recombinase Cre: FLPm

A transfer cassette encoding a Cre-FLPm chimeric recombinase was transformed into plant cells having an expression cassette encoding GUS driven by the ubiquitin promoter, in which a sequence flanked by either the FRT or loxP sites identical ones interrupted the GUS open reading frame. Figures 2 and 3 show that the Cre-FLPm chimeric recombinase is independently functional at either the FRT site or the loxP site, as measured by the ability to activate GUS activity after sequence cleavage between two target sites. identical, thereby bringing GUS activity under the control of the ubiquitin promoter.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to whom this invention is directed.

Although the foregoing invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, it will be obvious that certain changes and modifications can be made within the scope of the appended claims.