Modified frt recombination sites and methods of use

IS 2 390 132 T3

DESCRIPTION

Modified FRT recombination sites and methods of use.

Field of the invention

The invention relates to site-specific recombination systems and methods of use.

Background

Random insertion of introduced DNA into the genome of a host cell can be lethal if the foreign DNA turns out to insert into, and thus mutates, a critically important native gene. Furthermore, even if a random insertion event does not alter the function of a gene in a host cell, the expression of an inserted foreign nucleotide sequence can be influenced by positional effects caused by adjacent genomic DNA. In some cases, the nucleotide sequence is inserted at a site where the positional effect is strong enough to suppress the function or regulation of the introduced nucleotide sequence. In other cases, overproduction of the gene product has negative effects on a cell.

For example, in plants, positional effects can cause reduced agronomics, additional costs for additional research, creation of additional transgenic events, and slower production time. For these reasons, efficient methods are needed to direct the insertion of nucleotide sequences into the genome of various organisms, such as plants, at chromosomal positions that allow the desired function of the sequence of interest.

Schlake. T. and Bode, J. (Biochem. (1994) 33: 12746-51) describe the use of mutated FLP recognition sites (FRTs) for exchanging expression cassettes at defined chromosomal loci.

Summary

Methods and compositions are provided that use populations of randomized modified FRT recombination sites to identify, isolate and / or characterize modified FRT recombination sites. Recombinogenic modified FRT recombination sites are also provided that can be employed in a variety of methods for targeted recombination of polynucleotides of interest, including methods for recombining polynucleotides, evaluating promoter activity, directly selecting transformed organisms, minimizing or eliminating expression resulting from integration. randomize in the genome of an organism, such as a plant, remove polynucleotides of interest, combining multiple transfer cassettes, inverting or cleaving a polynucleotide, and identifying and / or characterizing transcriptional regulatory regions.

The invention provides an isolated polynucleotide comprising a nucleotide sequence comprising at least a first FRT recombination site comprising SEQ ID No. 21 and a second FRT recombination site comprising SEQ ID No. 39.

The invention further provides a cell comprising the polynucleotide of the invention.

The invention further provides a plant comprising a cell of the invention.

The invention further provides a method of directing the insertion of a polynucleotide of interest to a target site, said method comprising:

(a) providing a cell that has stably integrated into its genome the target site comprising a first recombination site comprising SEQ ID No. 21 and a second recombination site comprising SEQ ID No. 39;

(b) providing a transfer cassette comprising the polynucleotide of interest, wherein said polynucleotide of interest is flanked by said first and said second recombination sites; and (c) providing a FLP recombinase, wherein said recombinase recognizes and performs recombination at the first and second recombination sites, and the polynucleotide of interest is inserted into the target site.

Detailed description

The invention provides an isolated polynucleotide comprising a nucleotide sequence comprising a functional modified first FRT recombination site comprising the nucleotide sequence set forth in SEQ ID No. 21 and a second FRT combining site comprising SEQ ID No. 39.

Also provided are organisms, including, for example, prokaryotes, such as bacteria, and eukaryotes, such as yeast, plants, plant cells, and seeds that comprise the aforementioned polynucleotides comprising the modified FRT recombination sites. In specific examples, polynucleotides are integrated as

ES 2 390 132 T3 stable form in the genome of the organism.

A method of directing the insertion of a polynucleotide of interest is provided. The method comprises providing a target site having functional first and second recombination sites of the invention. A transfer cassette is provided comprising a polynucleotide of interest and the two functional recombination sites, where the second functional recombination site is recombinogenic with the first functional recombination site, and the first and / or second recombination site comprise a modified FRT site described in this document. At least one recombinase is provided. Recombinase recognizes and carries out recombination at the first and second recombination sites. The method can happen in vitro or in vivo. Functional recombination sites are stably integrated into the genome of a cell at the target site. In some examples, the polynucleotide of interest and / or the target can subsequently be cleaved, inverted, or otherwise modified, for example, by adding a second polynucleotide of interest to the target site.

The wild-type minimal FRT recombination site has been characterized and comprises a series of domains including the following nucleotide sequence 5'-AGTTCCTATTCTCTAGAAAGTATAGGAACT-3 '(SEQ ID NO: 39). The minimal FRT recombination site domains comprise a pair of 11 base pair symmetry elements that are the FLP binding sites (nucleotides 1-11 and 20-30 of SEQ ID NO: 39); the 8 base pair core, or spacer region (nucleotides 12-19 of SEQ ID NO: 39); and, the polypyrimidine extensions (nucleotides 3-14 and nucleotides 16-29 of SEQ ID No. 39). A modified FRT recombination site can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more alterations that include substitutions, additions, and / or deletions in one or more of these domains.

A modified FRT recombination site is provided. A modified FRT recombination site is a nucleotide sequence that is similar but not identical to the minimal native FRT recombination site set forth in SEQ ID NO: 39. The modified FRT recombination site retains the biological activity of the wild-type FRT recombination site and comprises a functional recombination site that is recognized by an FLP recombinase and capable of a recombinase-mediated recombination reaction. The modified FRT recombination site comprises a substitution of one or more nucleotides at one or more internal sites in the minimal native FRT recombination site. The modified FRT recombination site comprises SEQ ID NO: 21. Generally, modified recombination sites will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, sequence identity with the minimal native recombination site over its full length.

The modified FRT recombination site has nucleotide substitutions throughout the full length of the minimal recombination site.

The modified FRT recombination site has alterations in the 8 base pair spacer domain. The modified FRT12 spacer domain is set forth in SEQ ID NO: 1. The modified FRT site is functional. The modified FRT recombination site comprises the spacer region set forth in SEQ ID NO 1 and further comprises symmetry element FLP binding sites corresponding to those found in the minimal native FRT recombination site. See SEQ ID Nos. 19 and 20 showing wild-type symmetry element sequences. The modified FRT recombination site is set forth in SEQ ID NO: 21.

The modified FRT site comprising SEQ ID No. 21 is used in combination with the wild-type FRT site comprising SEQ ID No. 39 in a composition or method of the invention.

As discussed above, the modified recombination site is functional. A functional recombination site is a recombination site that is recombinogenic with a recombination site in the presence of the appropriate recombinase, and unless otherwise indicated, a recombination site is functional and includes wild-type sites, modified sites, variants , and fragments. Methods for determining whether a modified recombination site is recombinogenic are known. As used herein, a functional variant recombination site comprises a functional modified recombination site.

The recombination sites used in the methods are different sites. Different recombination sites, or a series of different recombination sites, are recombination sites that are distinct from each other because they have at least one nucleotide difference. Recombination sites within a number of different recombination sites can be recombinogenic or non-recombinogenic with respect to each other. Recombinogenic refers to recombination sites capable of recombining with one another. Unless otherwise noted, recombinogenic recombination sites or a series of recombination recombination sites include those sites where the relative recombination cleavage efficiency between the sites is greater than 2%, 5%, 10%, 20%. , 30%, 40%, 50%, 75%, 100%, or greater. As defined herein, the relative recombination cleavage efficiency is the cleavage efficiency in the presence of native recombinase of a modified first recombination site with a modified second recombination site divided by the cleavage efficiency of a pair of sites. native recombination rates X 100%. For example, when working with modified FRT sites, the relative recombination cleavage efficiency is defined as the cleavage efficiency in the presence of native FLP (SEQ ID NO: 49) of a modified first FRT site with a split modified second FRT site by

ES 2 390 132 T3 cleavage efficiency of a pair of native FRT sites (FRT1, SEQ ID NO: 39). Non-recombinogenic refers to recombination sites that in the presence of the appropriate recombinase will not recombine with one another, or recombination between the sites is minimal. Unless otherwise indicated, non-recombinogenic recombination sites, or a series of recombinogenic recombination sites, include those sites where the relative recombination cleavage efficiency between the sites is less than 2%, 1.5%, 1 %, 0.75%, 0.5%, 0.25%, 0.1%, 0.075, 0.005%, 0.001%.

The modified FRT recombination site comprising SEQ ID No. 21 is contained in a polynucleotide with an FRT site comprising SEQ ID No. 39. In one example, the polynucleotide comprises one or more expression units. An expression unit is a nucleotide sequence comprising a DNA unit characterized by having a single transcriptional promoter. Alternatively, the polynucleotide containing the modified FRT recombination site need not contain a promoter and / or downstream regulatory sequences. In other examples, the polynucleotide comprising the modified recombination site can be designed such that after integration into the genome, the sequences contained in the polynucleotide are functionally linked to an active promoter. It is recognized that a polynucleotide may have additional elements including, but not limited to, nucleotide sequences of interest, marker genes, recombination sites, termination regions, etc. As illustrated below, the polynucleotide can comprise transfer cassettes, target sites, or any part thereof.

An isolated or purified polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. An isolated or purified polynleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical agents when chemically synthesized. Typically, an isolated polynucleotide is free of sequences that naturally flank the 5 'and / or 3' ends of the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various examples, the isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the naturally flanking nucleotide sequence. the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes protein preparations that have less than about 30%, 20%, 10%, 5%, or 1% by dry weight of contaminating protein. When the biologically active protein itself is produced recombinantly, generally the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemical agents not of the protein of interest.

Polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include molecules of both natural origin and synthetic analogs. Polynucleotides also encompass all sequence forms including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

In one example, an isolated polynucleotide is provided, wherein the polynucleotide comprises the modified FRT recombination site comprising SEQ ID No. 21 and a FRT site comprising SEQ ID No. 39. In specific examples, the site of Modified FRT recombination is the polynucleotide sequence. In specific examples, the modified FRT recombination site is heterologous to the polynucleotide.

Heterologous refers to a polypeptide or nucleotide sequence that is native to a different species, or if it is of the same species, is substantially modified from its native form in composition and / or genomic locus. For example, a heterologous recombination site is a polynucleotide that is not found in the native polynucleotide or that is not in the same location in the native polynucleotide, and / or is modified from its native composition.

In other examples, an isolated polynucleotide is provided comprising a nucleotide sequence set forth in SEQ ID No. 21 and a nucleotide sequence set forth in SEQ ID No. 39.

A modified FRT recombination site can be introduced into an organism of interest. Introduction comprises presenting to the body at least one molecule, composition, polynucleotide, or polypeptide, such that the composition gains access to the interior of a cell. The methods do not depend on a particular method of introducing a polynucleotide or polypeptide into an organism, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the organism.

Organisms of interest include, but are not limited to, both prokaryotic and eukaryotic organisms including, for example, bacteria, yeast, and plants. In one example, The organism is a plant.

Methods for providing or introducing a composition into various organisms are known and include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and sexual reproduction. Stable transformation indicates that the introduced polynucleotide is integrated into the genome of the organism and is capable of being inherited by its offspring. Transient transformation indicates that the introduced composition is expressed or present only temporarily in the body.

IS 2 390 132 T3

Protocols for introducing polynucleotides and polypeptides into plants can vary depending on the type of plant or plant cell targeted for transformation, such as monocots or dicots. Suitable methods for introducing polynucleotides and polypeptides into plant cells and their subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334; and US patent 6,300,543), meristem transformation (US patent 5,736,369), electroporation (Riggs et al. (1986) Proc Natl Acad Sci USA 83: 5602-5606), Agrobacterium-mediated transformation (patents US 5,563,055; and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J 3: 2717-2722), and ballistic particle acceleration (US patents 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6: 923-926; Weissinger et al. (1988) Ann Rev Genet 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol 87: 671-674 (soybean); Finer and McMullen (1991) In Vitro Cell Dev Biol 27P: 175-182 (soy); Singh et al. (1998) Theor Appl Genet 96: 319-324 (soy); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc Natl Acad Sci USA 85: 4305-4309 (corn); Klein et al. (1988) Biotechnology 6: 559-563 (corn); US Patents 5,240,855; 5,322,783, and 5,324,646; Klein et al. (1988) Plant Physiol 91: 440-444 (corn); Fromm et al. (1990) Biotechnology 8: 833-839 (corn); HooykaasVan Slogteren et al. (1984) Nature 311: 763-764; US Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc Natl Acad Sci USA 84: 5345-5349 (Litiaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), p. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Rep 9: 415-418) and Kaeppler et al. (1992) Theor Appl Genet 84: 560-566 (filament-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Rep 12: 250-255; Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); and, Osjoda et al. (1996) Nat Biotechnol 14: 745-750 (maize by Agrobacterium tumefaciens).

Alternatively, the polynucleotides can be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide into a viral DNA or RNA molecule. It is recognized that a polypeptide of interest can be initially synthesized as part of a viral polyprotein, which can then be processed by in vivo or in vitro proteolysis to produce the desired recombinant protein. Furthermore, it is recognized that promoters also encompass promoters used for transcription of viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known, see, for example, US patents 5,889,191, 5,889,190, 5,866,785, 5,589 .367 and 5,316,931.

Transient transformation methods include, but are not limited to, the introduction of polypeptides, such as the recombinase protein, directly into the body, the introduction of polynucleotides such as DNA and / or RNA polynucleotides, and the introduction of the RNA transcript, such as an mRNA encoding a recombinase, in the body. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen Genet 202: 179-185; Nomura et al. (1986) Plant Sci 44: 53-58; Hepleret al. (1994) Proc Natl Acad Sci USA 91: 2178-2180; and, Hush et al. (1994) J Cell Sci 107: 775-784.

Cells having the introduced sequence can be grown in plants in conventional ways, see, for example, McCormick et al. (1986) Plant Cell Rep 5: 81-84. These plants can then be grown, and pollinated with the same transformed strain or with a different strain, and the resulting offspring that express the desired phenotypic trait and / or that comprise the introduced polynucleotide or polypeptide identified. Two or more generations can be grown to ensure that the polynucleotide is stably maintained and inherited, and the seeds harvested. Thus, the transformed seed, also referred to as a transgenic seed, is provided which has a polynucleotide, eg comprising a modified FRT site, stably incorporated into its genome.

Examples of plant genera and species of interest include, but are not limited to, monocots and dicots such as corn (Zea mays), Brassica sp. (for example, B. napus, B. rapa, B. júncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet (Pennisetum glaucum), common millet (Panicum miliaceum), lesser millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max) , tobacco (Nicotiana tabacum), potato (Solanum tuberosum), groundnut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot escuienta), coffee (Coffea spp.), coconut (Cocos nucífera), pineapple (Ananas comosus) ), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus caslca), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europae), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oats (Avena), barley (Hordeum), palm, legumes including beans and peas such as guar, carob, fenugreek, beans, cowpea, Chinese bean, Lima bean, broad bean, lentils, chickpea, and castor bean, Arabidopsis, vegetables, ornamental plants, herbs, conifers; crop and grain plants that provide seeds of interest, oilseeds, and other legume plants. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), lima beans

ES 2 390 132 T3 (Phaseolus limensis), peas (Lathyrus spp.), And members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and cantaloupe (C. melo). Ornamental plants include azalea (Rhododendron spp.), Hydrangea (Macrophylla hidrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers include, for example, pines such as taeda pine (Pinus taeda), ellioti pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), contorta pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); sitka fir (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsemea); and cedars such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis).

The term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calluses, plant mats, and plant cells that are intact in plants or plant parts such as embryos, pollen, ovules, seeds, flowers, bones, spikes, ears, husks, stems, roots, root tips, anthers, and the like.

Prokaryotic cells can also be used in the methods. Prokaryotes include various strains of E. coli; however, other microbial strains can also be used including, for example, Bacillus sp, Salmonella, and Agrobacterium. Exemplary Agrobacterium strains include c58c1 (pGUSINT), Agt121 (pBUSINT), EHA101 (pMTCA23GUSINT), EHA105 (pMT1), LBA4404 (pTOK233), GU2260, BU3600, AGL-1, and LBA4402. Said strains are described in detail in Chan et al. (1992) Plant Cell Physiol 33: 577; Smith et al. (1995) Crop Sci 35: 301; and Hiei et al. (1994) Plant J 6: 271-282. Exemplary bacterial strains include, but are not limited to, C600 (ATCC 23724), C600hfl, DH1 (ATCC 33849), DH5a, DH5aF ', ER1727, GM31, GM119 (ATCC 53339), GM2163, HB101 (ATCC 33694), JM83 (ATCC 35607 ), JM101 (ATCC 33876), JM103 (ATCC 39403), JM105 (ATCC 47016), JM107 (ATCC 47014), JM108, JM109 (ATCC 53323), JM110 (ATCC 47013), LE392 (ATCC 33572), K802 (ATCC 33572) ), NM522 (ATCC 47000), RR1 (ATCC 31343), X1997 (ATCC 31244), and Y1088 (ATCC 37195). See also, Jendrisak et al. (1987) Guide to Molecular Cloning Techniques, Academic Press, 359-371, Hanahan et al. (1983) J Mol Biol 166: 557580, Schatz et al. (1989) Cell 59: 1035, Bullock et al. (1987) Bio-Techniques 5: 376-378, ATCC Bacteria and Bacteriophages (1996) 9^to Edition, and Palmer et al. (1994) Gene 143: 7-8.

Exemplary but not limiting viral strains include, but are not limited to, geminivirus, begomovirus, curtovirus, mastrevirus, RNA strand virus (-), RNA strand virus (+), potyvirus, potexvirus, tobamovirus, or other DNA virus, nanovirus, viroid , and the like, for example, African cassava mosaic virus (ACMV) (Ward et al (1988) EMBO J 7: 899-904 and Hayes et al. (1988) Nature 334: 179-182), mosaic virus barley streaky (BSM) (Joshi et al. (1990) EMBO J 9: 2663-2669), Cauliflower mosaic virus (CaMV) (Gronenborn et al. (1981) Nature 294: 773-776 and Brisson et al. (1984) Nature 310: 511-514) , maize streak virus (MSV) (Lazarowitz et al. (1989) EMBO J 8: 1023-1032 and Shen et al. (1994) J Gen Virol 76: 965-969), tobacco mosaic virus (TMV ) (Takamatsu et al. (1987) EMbO J 6: 307-311 and Dawson et al. (1989) Virology 172: 285-292), tomato gold mosaic virus (TGmV) (Elmer et al. (1990) Nucleic Acids Res 18: 2001-2006), and wheat dwarf virus (WDV) (Woolston et al. (1989) Nucleic Acids Res 17: 6029-6041) and derivatives thereof. See also, Porat et al. (1996) Mol Biotechnol 5: 209-221.

Commonly used prokaryotic control sequences include promoters for the initiation of transcription, optionally with an operator, along with ribosome binding sequences, include commonly used promoters such as the beta lactamase (penicillinase) and lactose (lac ) (Chang et al. (1977) Nature 198: 1056), the tryptophan (trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res 8: 4057) and the lambda-derived PL promoter and ribosome binding site of the N gene (Shimatake et al. (1981) Nature 292: 128).

The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Prokaryotic / bacterial expression systems are available for the expression of a protein using Bacillus sp. and Salmonella (Palva et al. (1983) Gene 22: 229-235; Mosbach et al. (1983) Nature 302: 543-545). The Tet operon and Lac operon can also be used.

A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells are known for the expression of a polynucleotide of interest. In some examples, transformed / transfected plant cells are used as expression systems. The synthesis (introduction / expression) of heterologous nucleotide sequences in yeast is well known (Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory). Two yeasts widely used for the production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known and available from commercial providers (eg, InVitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences, and the like, as desired.

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Recombinant baculoviruses are generated by inserting the sequences of particular interest into the baculovirus genome using established protocols with vectors and reagents from commercial vendors (eg, InVitrogen, Life Technologies Incorporated). Commercial vectors are readily available with various promoters, such as polyhedrin and p10, optional signal sequences for protein secretion, or affinity tags, such as 6X histidine. These recombinant viruses are grown, maintained, and propagated in commercially available cell lines derived from various insect species including Spodoptera frugiperda and Trichoplusla nl. Insect cells can be cultured using well-established protocols in a variety of different media, for example, with and without bovine serum supplementation. The cultured cells are infected with the recombinant viruses and the sequence of interest is expressed. Proteins expressed with the baculovirus system have been extensively characterized and, in many cases, their post-translational modifications such as phosphorylation, acylation, etc., are identical to the natively expressed protein.

The modified FRT recombination site of the invention, in combination with FRT1 can be used as reagents in kits. Such kits may further comprise a FLP recombinase, and may further comprise a polynucleotide, optionally integrated into the genome of an organism, having at least one target site flanked by a different functional, non-recombinogenic, modified FRT recombination site. Any kit can be additionally accompanied by instructions for use.

Recombinogenic modified FRT recombination sites can be used in various in vitro and in vivo site-specific recombination methods that allow for the integration, exchange, modification, alteration, cleavage, inversion, and / or targeted expression of a nucleotide sequence of interest, see, for example, WO99 / ​​25821, WO99 / ​​25854, WO99 / ​​25840, WO99 / ​​25855, and WO99 / ​​25853.

The methods employ a site-specific recombination system. A site-specific recombinase, also referred to as a recombinase, is a polypeptide that catalyzes conservative site-specific recombination between its compatible recombination sites, therefore a recombinase includes native polypeptides as well as variants and / or fragments that retain activity, and native polynucleotides and variants and / or fragments that encode a recombinase that retains activity. The recombinase used in the methods can be a native recombinase or a biologically active or variant fragment of the recombinase. A native polypeptide or polynucleotide comprises a naturally occurring amino acid sequence or nucleotide sequence. For reviews of site-specific recombinases, see Sauer (1994) Curr Op Biotechnol 5: 521-527; and Sadowski (1993) FASEB 7: 760-767. Recombinases useful in the methods and compositions include recombinases of the Integrase and Resolvasse families, biologically active variants and fragments thereof, and any other naturally occurring or recombinantly produced enzyme or variant thereof, which catalyzes specific conservative recombination. of site between specific DNA recombination sites.

The Integrase family of recombinases has more than one hundred members and includes, for example, FLP, Cre, integrase lambda, and R. For other members of the Integrase family see, for example, Esposito et al. (1997) Nucleic Acids Res 25: 3605-3614 and Abremski et al. (1992) Protein Eng 5: 87-91. Other recombination systems include, for example, streptomycetes bacteriophage phi C31 (Kuhstoss et al. (1991) J Mol Biol 20: 897-908); the Sulfolobus shibatae SSV1 site-specific recombination system (Maskhelishvili et al. (1993) Mol Gen Genet 237: 334342); and an integration system based on retroviral integrase (Tanaka et al. (1998) Gene 17: 67-76). Some recombinases do not require cofactors or a supercoiled substrate. Said recombinases include native Cre (SEQ ID N ° 45 and 46), native FLP (SEQ ID N ° 48 and 49), or active variants or fragments thereof (SEQ ID N ° 47 and 50).

FLP recombinase is used in the method of the invention. FLP recombinase is a protein that catalyzes a site-specific reaction that is involved in amplifying the number of copies of the two-micron plasmid of S. cerevisiae during DNA replication. FLP recombinase catalyzes site-specific recombination between two FRT sites. The FLP protein has been cloned and expressed (Cox (1993) Proc Natl Acad Sci USA 80: 4223-4227). The FLP recombinase for use in the methods and with the compositions can be obtained from the genus Saccharomyces. A polynucleotide comprising the recombinase can also be synthesized using plant-preferred codons for optimal expression in a plant of interest. A recombinant FLP enzyme encoded by a nucleotide sequence comprising corn preferred codons (mFLP) is known that catalyzes site-specific recombination events (SEQ ID No. 50, and US Patent 5,929,301). Additional functional variants and fragments of FLP are known (Buchholz et al. (1998) Nat Biotechnol 16: 617-618, Hartung et al. (1998) J Biol Chem 273: 22884-22891, Saxena et al. (1997) Biochim Biophys Acta 1340: 187-204, and Hartley et al. (1980) Nature 286: 860-864).

Bacteriophage Cre recombinase catalyzes site-specific recombination between two lox sites. Cre recombinase is known (Guo et al. (1997) Nature 389: 40-46; Abremski et al. (1984) J Biol Chem 259: 15091514; Chen et al. (1996) Somat Cell Mol Genet 22: 477-488 ; Shaikh et al. (1977) J Biol Chem 272: 5695-5702; and, Buchholz et al. (1998) Nat Biotechnal 16: 617-618). Cre polynucleotide sequences can also be synthesized using plant-preferred codons, for example such sequences (moCre) are described in WO 99/25840 and set forth in SEQ ID NO: 47.

IS 2 390 132 T3

A chimeric recombinase is a recombinant fusion protein that is capable of catalyzing site-specific recombination between recombination sites originating from different recombination systems. Methods for the production and use of such chimeric recombinases or variants or active fragments thereof are described in WO 99/25840. Assays for recombinase activity are known and generally measure the overall activity of the enzyme on DNA substrates containing recombination sites. For example, to test for FLP activity, inversion of a DNA sequence in a circular plasmid containing two inverted FRT sites can be detected as a change in the position of the restriction enzyme sites. This assay is described in Vetter et al. (1983) PNAS 80: 7284. Alternatively, the DNA cleavage of a molecule or the frequency of intermolecular recombination induced by the enzyme can be tested, as described, for example, in Babineau et al. (1985) J Biol Chem 260: 12313; Meyer-Leon et al. (1987) Nucleic Acid Res 15: 6469; and Gronostajski et al. (1985) J Biol Chem 260: 12328. Alternatively, recombinase activity can also be assayed by cleavage of a sequence flanked by recombinogenic FRT sites that upon removal will activate an assayable marker gene. Similar assay strategies can be used for Cre or other recombinase enzymes.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and / or recombinogenic procedure such as DNA dragging. With such a method, one or more different recombinase coding sequences can be manipulated to create a new recombinase protein having the desired properties. Thus, recombinant polynucleotide libraries are generated from a population of related polynucleotides comprising regions of sequence that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA carry-over are known and include, for example, Stemmer (1994) Proc Natl Acad Sci USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nat Biotech 15: 436-438; Moore et al. (1997) J Mol Biol 272: 336-347; Zhang et al. (1997) Proc Natl Acad Sci USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and US Patents 5,605,793 and 5,837,458.

The methods and compositions employ the modified FRT sites provided herein, FRT12 (SEQ ID No. 21), in combination with FRT1 (SEQ ID No. 39). A recombination site is any native or synthetic / artificial polynucleotide that is recognized by the recombinase enzyme of interest. Many recombination systems are known as well as the appropriate recombination site (s) for use with the recombination system of interest, including biologically active variants and fragments of recombination sites. Additional recombination sites can be used with the FRT12 / FRT1 combination of the invention. Examples of recombination sites for use are known and include FRT sites including the native FRT site (FRT1, SEQ ID NO: 39), and various functional variants of FRT, including, but not limited to, FRT5 (SEQ ID NO: 40). , FRT6 (SEQ ID No. 41), FRT7 (SEQ ID No. 42), FRT87 (SEQ ID No. 24), and the other functional modified FRT sites. See, for example, WO 03/054189, WO 02/00900, WO 01/23545, and Schlake et al. (1994) Biochemistry 33: 12745-12751.

Recombination sites of the Cre / Lox site-specific recombination system can also be used in addition to the FRT12 / FRT1 combination of the invention. Such recombination sites include, for example, native LOX sites and various functional LOX variants. An analysis of the recombination activity of variant LOX sites is presented in Lee et al. (1998) Gene 216: 55-65 and in US Patent 6,465,254. See also, for example, Schlake and Bode (1994) Biochemistry 33: 12746-12751; Huang et al. (1991) Nucleic Acids Res 19: 443448; Sadowski (1995) In Progress in Nucleic Acid Research and Molecular Biology Vol. 51, p. 53-91; US Patent 6,465,254; Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society of Microbiology, Washington DC, p. 116-670; Dixon et al. (1995) Mol Microbiol 18: 449-458; Umlauf and Cox (1988) EMBO J 7: 18451852; Buchholz et al. (1996) Nucleic Acids Res 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) Plant J 7: 649-659; Bayley et al. (1992) Plant Mol Biol 18: 353361; Odell et al. (1990) Mol Gen Genet 223: 369-378; Dale and Ow (1991) Proc Natl Acad Sci USA 88: 10558-10562; Qui et al. (1994) Proc Natl Acad Sci USA 91: 1706-1710; Stuurman et al. (1996) Plant Mol Biol 32: 901-913; Dale et al. (1990) Gene 91: 79-85; and WO 01/111058.

Any suitable recombination site or series of recombination sites can be used in methods and compositions in addition to the FRT12 / FRT1 combination of the invention, including a FRT site, a functional variant of a FRT site, a LOX site, and a functional variant of a LOX site, any combination thereof, or any other combination of known recombination sites.

Directly repeating indicates that the recombination sites in a series of recombinogenic recombination sites are arranged in the same orientation, such that recombination between these sites causes cleavage, rather than inversion, of the intermediate DNA sequence. Inverted recombination site (s) indicates that the recombination sites in a series of recombinogenic recombination sites are arranged in the opposite orientation, so that recombination between these sites causes inversion, rather than cleavage, of the intermediate DNA sequence .

The target site and the transfer cassette used in the method of the invention comprise a first recombination site comprising SEQ ID No. 21 and a second recombination site comprising SEQ ID No.

IS 2 390 132 T3

39. The site-specific recombinase used will be a FLP recombinase or an active variant thereof will be provided. Likewise, when Lox sites are also used, a Cre recombinase or active variant thereof can be used as well. If the series of functional recombination sites comprises both FRT12 and FRT1 sites and a Lox site, a chimeric FLP / Cre recombinase or an active variant or both FLP and Cre recombinases or active variants thereof can be used.

Providing includes any method that allows a polypeptide and / or a polynucleotide such as a recombinase, target site, transfer cassette, polynucleotide of interest to be assembled with the indicated components. For example, a cell can be provided with these various components by a variety of methods including transient and stable transformation methods; co-introducing a DNA, mRNA, or protein recombinase directly into the cell; employing an organism, cell, strain or line that expresses the recombinase for initial transformation; or by growing / culturing the organism that carries the target site and crossing it with an organism that expresses an active recombinase protein and selecting for events in the offspring. Any promoter can be used, including a constitutive, inducible, developmental / temporal regulated, or spatially regulated promoter capable of regulating expression in the organism of interest to express the appropriate recombinase.

Compositions comprising a modified recombinogenic FRT recombination site are provided. The polynucleotide of the invention, comprising the recombinogenic modified FRT recombination site FRT12 (SEQ ID No. 21) and wild-type FRT1 (SEQ ID No. 39) can be used in site-specific recombination methods.

The methods can employ target sites and transfer cassettes to allow manipulation, exchange, cleavage, alteration, inversion, and / or introduction of a nucleotide sequence in vivo or in vitro. A target site comprises at least one recombination site. In specific examples, the target site comprises a polynucleotide that is immediately flanked by at least two recombination sites, including series of functional recombination sites that are different and non-recombinogenic with respect to one another; corresponding and recombinogenic with respect to each other; or different and recombinogenic with respect to each other. One or more intermediate sequences may be present between the recombination sites of the target site. Intermediate sequences of particular interest include linkers, adapters, regulatory regions, introns, restriction sites, enhancers, isolators, selection markers, nucleotide sequences of interest, promoters, and / or other sites that aid vector construction or analysis. . It is further recognized that a recombination site may be contained within the nucleotide sequence of interest including introns, coding sequence, 5 'UTR, 3' UTR, and / or regulatory regions.

In specific examples, the target site is on a cell or organism of interest. In other examples, the target site is stably integrated into the genome of the cell or organism of interest. It is recognized that the cell or organism may comprise multiple target sites, which may be located at one or multiple loci within or throughout the chromosomes. Multiple independent manipulations of each target site in the body are available. Furthermore, the target site may also comprise an expression cassette comprising a nucleotide sequence encoding an appropriate recombinase. In another example, the nucleotide sequence encoding the recombinase is stably integrated into the genome of the organism.

The methods additionally employ transfer cassettes. A transfer cassette comprises at least one recombination site. In specific examples, the transfer cassette comprises a polynucleotide flanked by at least a first recombination site and a second recombination site, where the first and second recombination sites correspond to the recombination sites at the target site. The first and second functional recombination sites of the transfer cassette may be different and non-recombinogenic with respect to each other. When a target site and a transfer cassette comprising compatible recombination sites and the recombinase are combined, the nucleotide sequence between the recombination sites of the target site will be exchanged with the nucleotide sequence between the recombination sites of the transfer cassette. Flanked by, when used in reference to the position of the recombination sites of the target site or the transfer cassette, refers to a position immediately adjacent to the intended sequence to be exchanged or inserted.

The transfer cassette may further comprise a polynucleotide of interest. The recombination sites may be directly contiguous to the polynucleotide of interest or there may be one or more intervening sequences present between one or both ends of the polynucleotide of interest and the recombination sites. Intermediate sequences of particular interest include linkers, adapters, enhancers, introns, isolators, restriction sites, selection markers, polynucleotides of interest, promoters, and / or other sites that aid in vector construction or analysis. Recombination sites can be contained within the polynucleotide of interest including within introns, coding sequence, and / or 5 'and 3' untranslated regions.

In the method of the invention, the transfer cassette and the target site comprise a functional modified FRT recombination site comprising SEQ ID No. 21 and a second recombination site comprising SEQ ID No. 39.

IS 2 390 132 T3

Any means can be used to bring together the various components of the recombination system. For example, in in vitro systems, the recombinase and the polynucleotide (s) comprising the recombination sites can be provided by contacting the components under appropriate conditions to allow a recombination event. Alternatively, a variety of methods are known for the introduction of nucleotide and polypeptide sequences into an organism including, for example, transformation, and introduction of the polypeptide, DNA, or mRNA into the cell. See also WO99 / ​​25884.

The methods find use in various applications. For example, the methods may employ the use of two modified functional FRT recombination sites and allow in vivo and in vitro exchange, insertion, inversion, or cleavage of a nucleotide sequence of interest. For example, the cell or organism of interest may comprise a first polynucleotide comprising a target site comprising a functional modified first FRT recombination site. The cell or organism is provided with a second polynucleotide comprising a transfer cassette comprising a corresponding and functional second FRT recombination site or a different second FRT site that is recombinogenic with respect to the first site. An FLP recombinase is provided under conditions that allow for a recombination event. The recombination event between the two recombinogenic recombination sites causes the insertion of the transfer cassette together with the second complete polynucleotide that is contained in the first polynucleotide. In some examples, the first polynucleotide is stably integrated into the genome of the organism. The method can also be used in an in vitro context. For example, the first and second polynucleotides can comprise polynucleotides such as plasmids combined in vitro in the presence of an appropriate recombinase. In this example, a recombination event will produce a co-integrating plasmid. Such methods find use, for example, in various cloning technologies, including PCR amplification of fragments (Sadowski et al. (2003) BMC Biotechnol 18: 9), cloning vectors (Snaith et al. (1995) Gene 166: 173-174 and US patents 6,140,129, 6,410,317, 6,355,412, 5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608) and viral vectors (US Patent 6,541,245).

In other examples, the method comprises providing a target site having a functional first and second recombination site, where the first and second recombination sites are different and non-recombinogenic with respect to each other and at least one of the first or second. recombination sites comprise a functional modified FRT recombination site described herein; providing a transfer cassette comprising a polynucleotide of interest flanked by the first and second recombination sites; and, providing a recombinase. Recombinase recognizes and carries out recombination at the first and second recombination sites.

In specific examples, the target site is on a host cell or organism; and, in other examples, the target site is stably integrated into the genome of the host cell or organism. In still other examples, the target site comprises a polynucleotide of interest. In this example, if the target site and the transfer cassette comprise the first and second recombination sites that are different and non-recombinogenic with respect to each other, the sequence of interest in the target site is exchanged for a second contained polynucleotide of interest. in the transfer cassette.

In some examples, multiple promoters can be used to regulate transcription at a single target site. In this case, the target site comprising the first and second recombination sites is flanked by two convergent promoters. Convergent promoters refers to promoters that are oriented at either end of the target site. The same promoter, or different promoters can be used at the target site. Each of the convergent promoters is operably linked to either the first or the second recombination site. For example, the target site flanked by convergent promoters may comprise P1 ^: R1R2: —P2, where P is a promoter, the date indicates the direction of transcription, R is a recombination site, and the colon indicates that the components they are functionally linked.

The transfer cassette used with the target site having the convergent promoters may comprise, in the following order, the first recombination site, a first polynucleotide of interest oriented in the 5 'to 3' direction, a second polynucleotide of interest oriented in the 3 'to 5' direction, and a second recombination site. Insertion of the transfer cassette into the target site causes the first polynucleotide of interest to be operably linked to the first convergent promoter, and the second polynucleotide of interest to be operably linked to the second convergent promoter. The expression of the first and / or second polynucleotide of interest can be increased or decreased in the cell or organism. The expression of the first and / or second polynucleotide of interest can also be regulated independently depending on the promoters that are used. It is recognized that the target sites may be flanked by other elements that influence transcription. For example, insulating elements can flank the target site to minimize positional effects. See, for example, US Publication No. 2005/0144665.

Any promoter can be used, and is typically selected based on the desired result. A promoter is a region of DNA involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A plant promoter is a promoter capable of initiating transcription in a plant cell, to

ES 2 390 132 T3 a review of plant promoters see Potenza et al. (2004) In Vitro Cell Dev Biol 40: 1-22.

Constitutive promoters include, for example, the minimal promoter of the Rsyn7 promoter and other constitutive promoters described in WO 99/43838 and US Patent 6,072,050; the CaMV 35S minimal promoter (Odell et al. (1985) Nature 313: 810-812); from 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: 675-689); pEMU (Last et al. (1991) Theor Appl Genet 81: 581-588); MAS (Velten et al (1984) EMBO J 3: 27232730); ALS (US Patent 5,659,026), and the like. Other constitutive promoters are described in, for example, US Patents 5,608,149; 5,608,144; 5,604,121; 5,669,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In some examples, an inducible promoter can be used. Pathogen-inducible promoters induced after infection by a pathogen include, but are not limited to, those that regulate the expression of PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfl et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Virol 4: 111-116; WO 99/43819; Marineau et al. (1987) Plant Mol Biol 9: 335-342; Matton et al. (1989) Mol Plant-Microbe Interact 2: 325-331; Somsisc h et al. (1986) Proc Natl Acad Sci USA 83: 2427-2430; Somsisch et al. (1988) Mol Gen Genet 2: 93-98; Yang (1996) Proc Natl Acad Sci USA 93: 14972-14977; Chen et al. (1996) Plant J 10: 955-966; Zhang et al. (1994) Proc Natl Acad Sci USA 91: 2507-2511; Warner et al. (1993) Plant J 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968; US Patent No. 5,750,386 (inducible by nematodes); and the references cited therein; and Cordero et al. (1992) Physiol Mol Plant Path 41: 189-200 (Fusarium inducible). Wound-inducible promoters include the potato proteinase inhibitor gene (pin II) (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotechnol 14: 494-498); wun1 and wun2 (US Patent 5,428,148); win1 and win2 (Stanford et al. (1989) Mol Gen Genet 215: 200-208); sistemin (McGurl et al. (1992) Science 225: 15701573); WIP1 (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Lett 323: 73-76); MPI gene (Corderok et al. (1994) Plant J 6: 141-150); and the like. Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter can be a promoter inducible by chemical agents, where the application of the chemical agent induces gene expression, or a promoter repressible by chemical agents, where the application of the chemical agent represses gene expression. Chemical inducible promoters include, but are not limited to, the maize In2-2 promoter, activated by benzenesulfonamide herbicide protectants (De Veylder et al. (1997) Plant Cell Physiol 38: 568-77), the corn GST promoter (GST-ll-27, WO 93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the PR-1a promoter tobacco (Ono et al. (2004) Biosci Biotechnol Biochem 68: 803-7) activated by salicylic acid. Other chemically regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc Natl Acad Sci USA 88: 10421-10425; and McNellis et al. (1998) Plant J 14: 247-257); tetracycline-inducible and tetracycline-repressible promoters (Gatz et al. (1991) Mol Gen Genet 227: 229-237; US Patents 5,814,618, and 5,789,156).

Tissue-preferred promoters can be used to drive enhanced expression of a sequence of interest within a particular plant tissue. Tissue-preferred promoters include Kawamata et al. (1997) Plant Cell Physiol 38: 792-803; Hansen et al. (1997) Mol Gen Genet 254: 337-343; Russell et al. (1997) Transgenic Res 6: 157-168; Rinehart et al. (1996) Plant Physiol 112: 1331-1341; Van Camp et al. (1996) Plant Physiol 112: 525-535; Canevascini et al. (1996) Plant Physiol 112: 513-524; Lam (1994) Results Probl Cell Differ 20: 181-196; and GuevaraGarcia et al. (1993) Plant J 4: 495-505.

Leaf-preferred promoters are known and include, for example, Yamamoto et al. (1997) Plant J 12: 255-265; Kwon et al. (1994) Plant Physiol 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol 35: 773-778; Gotor et al. (1993) Plant J 3: 509-18; Orozco et al. (1993) Plant Mol Biol 23: 1129-1138; Matsuoka et al. (1993) Proc Natl Acad Sci USA 90 (20): 9586-9590; and cab and rubisco promoters (Simpson et al. (1958) EMBO J 4: 2723-2729; Timko et al. (1988) Nature 318: 57-58).

Root-preferred promoters are known and include, for example, Hire et al. (1992) Plant Mol Biol 20: 207-218 (soybean root glutamine synthase gene); Miao et al. (1991) Plant Cell 3: 11-22 (cytosolic glutamine synthase (GS) expressed in soybean roots and root nodules; Keller and Baumgartner (1991) Plant Cell 3: 1051-1061 (root-specific control element in the GRP gene 1.8 of green beans); Sanger et al. (1990) Plant Mol Biol 14: 433-443 (A. tumefaciens mannopine synthase root specific promoter (MAS)); Bogusz et al. (1990) Plant Cell 2: 633-641 (root specific promoters isolated from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi (1991) Plant Sci 79: 69-76 (roIC and roID root inducer genes from A. rhizogenes); Teeri et al. (1989) EMBO J 8: 343-350 (Agrobacterium wound-induced TR1 'and TR2' genes); promoter of the VfENOD-GRP3 gene (Kuster et al. (1995) Plant Mol Biol 29: 759-772); and rolB promoter (Capana et al. (1994) Plant Mol Biol 25 (4): 681-691); phaseolin gene (Murai et al. (1983) Science 23: 476-482; Sengopta-Gopalen et al. (1988) Proc Natl Acad Sci USA 82: 3320-3324). See also US Patents 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

Preferred seed promoters include both seed-specific promoters active during the

ES 2 390 132 T3 seed development, as well as active seed germination promoters during seed germination. See, Thompson et al. (1989) BioEssays 10: 108. Preferred seed promoters include, but are not limited to, Cim1 (cytokine-induced message); cZ19B1 (19 kDa maize zein); and milps (myo-inositol-1 phosphate synthase); (see WO 00/11177 and US Patent 6,225,529). For dicots, preferred seed promoters include, but are not limited to, bean β-phaseolin, napine, βconglycinin, soybean lectin, cruciferin, and the like. For monocots, preferred seed promoters include, but are not limited to, corn 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrink 1, shrink 2, globulin 1, oleosin, and nuc1. See also WO 00/12733, where seed-preferred promoters of the endl and end2 genes are described.

In other examples, the target site is constructed to have multiple functional sets of different and non-recombinogenic recombination sites, including FRT12 (SEQ ID No. 21) / FRT1 (SEQ ID No. 39) of the invention. Thus, multiple genes or polynucleotides can be stacked or arranged. In specific examples, this method allows the stacking of sequences of interest at precise locations in the genome of a cell or organism. Also, once a target site has been established within a cell or organism, additional recombination sites can be introduced by incorporating those sites into the transfer cassette. Thus, once a target site has been established, it is possible subsequently to add sites or alter sites through recombination. Such methods are described in detail in WO 99/25821.

Recombination site conversion can also be employed to stack various polynucleotides in the genome of an organism, such as a plant. For example, in vivo conversion of recombination sites to create new sites, rather than reintroducing new recombination sites into the body. For example, the conversion of different, non-recombinogenic recombination sites flanking a selection marker to corresponding recombination sites would facilitate the removal of a selection marker, or allow the reuse of the same selection marker in future transformations, providing a means for recycle selection markers. A different recombination site with a known recombination frequency could also be modified in situ into a different recombination site with a similar or altered recombination frequency. Other modifications can be achieved to alter function, similarity, or recombinogenicity.

In another example, a plurality of copies of the polynucleotide of interest is provided to the organism, such as a plant. In some examples, this is achieved by incorporation of an extrachromosomal replicon into the transfer cassette (see WO 99/25855). In specific examples, the transfer cassette comprises a replicon and a polynucleotide of interest flanked by a directly repeated first and second recombination site of the invention, where the recombination sites are recombinogenic with respect to each other. When an appropriate recombinase is used, the transfer cassette flanked by the directly repeated first and second recombination sites is excised from the genome of the organism, eg a plant, producing a viable replicon containing the polynucleotide of interest. Replicon of this replicon produces a larger number of copies of the replicon, the polynucleotide of interest, and / or prolongs the availability of the transfer cassette within the cell. A third functional recombination site may be present between the replicon and the polynucleotide of interest, where the third and first recombination sites are functional and different and non-recombinogenic sites with respect to each other, and the presence of the appropriate recombinase allows for the integration of the polynucleotide of interest into a target site flanked by the third and first recombination sites.

A replicon comprises a self-replicating extrachromosomal unit. The replicon can originate from a virus, plasmid, or cell and has the ability to self-replicate. In this example, the transfer cassette used in the method of the invention can comprise both a replicon and the polynucleotide of interest. A transfer cassette used in the method of the invention may comprise in a 5 'to 3' or 3 'to 5' orientation: a first functional recombination site (FRT12; SEQ ID NO: 21), a replicon, a second site functional recombination (FRT1; SEQ ID NO: 39), the polynucleotide of interest, and a third functional recombination site. The first and third recombination sites of this transfer cassette are directly repeated, corresponding, and recombinogenic with respect to each other, and the second recombination site is different and non-recombinogenic with respect to the first and third recombination sites. The transfer cassette can be contained in a T DNA. The replicon can be a viral replicon. A viral replicon is any DNA or RNA derived from a virus that undergoes episomal replication in a host cell. It contains cis-acting viral sequences necessary for replication, eg, the origin of replication. It may or may not contain trans-acting viral sequences necessary for replication. The cleaved viral DNA is capable of acting as a replicon or replication intermediate, independently, or with trans-supplied factors. Viral DNA may or may not encode infectious viral particles and may also contain insertions, deletions, substitutions, rearrangements, or other modifications. Viral DNA can contain heterologous DNA. In this case, heterologous DNA refers to any non-viral DNA or DNA from a different virus. For example, the heterologous DNA can comprise an expression cassette for a protein or RNA of interest.

Viral replicons suitable for use in the methods and compositions include those from geminiviruses, begomoviruses, curtoviruses, or mastreviruses, RNA strand viruses (-), RNA strand viruses (+), potyviruses, potexviruses, and tobamoviruses. Viral replicons may also include those from viruses that have a circular DNA replication genome or intermediate, such as: Abuitilon mosaic virus (AbMV), African mosaic virus of

ES 2 390 132 T3 cassava (ACMV), banana streak virus (BSV), bean dwarf mosaic virus (BDMV), bean golden mosaic virus (BGMV), beet bite virus (BCTV), western beet yellow virus (BWYV) and other luteoviruses, latent cassava virus (CLV), carnation corrosion virus (CERV), cauliflower mosaic virus (CaMV), striated chloris mosaic virus (CSMV), commelin yellow spot virus (CoYMV), Cucumber mosaic virus (CMV), dahlia mosaic virus (DaMV), digitaria streak virus (DSV), celedonia mosaic virus (FMV), hop dwarfism viroid (HSV), maize streak (MSV), mirabilia mosaic virus (MMV), miscanthus streak virus (MiSV), potato dwarf tuber virus (PSTV), panicum streak virus (PSV), potato yellow mosaic (PYMV), potato virus X (PVX), Rice tungro bacilliform virus (RTBV), soybean chlorotic speckle virus (SoyCMV), squash leaf curl virus (SqLCV), strawberry vein band virus (SVBV), sugarcane (SSV), thistle mottle virus (ThMV), tobacco mosaic virus (TMV), tomato gold mosaic virus (TGMV), tomato mottle virus (TMoV), ring spot virus tobacco (TobRV), Tobacco Yellow Dwarf Virus (TobYDV), Tomato Leaf Curl Virus (TLCV), Tomato Yellow Leaf Curl Virus (TYLCV), Tomato Yellow Leaf Curl Virus - Thailand (TYLCV-t) and Tomato Leaf Curl Virus (TYLCV-t) wheat (WDV) and derivatives thereof. In some examples, the viral replicon can be from ACMV, MSV, WDV, TGMV, or TMV.

Insertion of a polynucleotide of interest into the genome of the organism can occur through a single crossover event. For example, the transfer cassette used in the method of the invention may comprise a first recombination site (FRT12; SEQ ID NO: 21), a replicon, a polynucleotide of interest, and a second recombination site (FRT1; SEQ ID NO. No. 39). The first and second recombination sites of the transfer cassette are recombinogenic, different or corresponding, and directly repeated with respect to each other. The target site may comprise a single recombination site that is recombinogenic with one of the recombination sites of the transfer cassette.

The transfer cassette is introduced into the organism that comprises the target site. When an appropriate recombinase is provided, a recombination event occurs between the recombinogenic recombination sites of the transfer cassette. This event causes the replicon to split, which can assume a circularized shape. Replicon unit replication causes a high number of replicon copies in the body and prolongs the availability of the transfer cassette in the cell. A second recombination event between the recombinogenic recombination sites of the target site and the transfer cassette allows stable integration of the replicon unit and the polynucleotide of interest into the target site of the organism.

The invention provides a method for the targeted insertion of a polynucleotide of interest. If the polynucleotide of interest is introduced into an organism, it can confer various changes in the organism, particularly plants, including, but not limited to, modification of the fatty acid composition in the plant, alteration of the amino acid content of the plant, the alteration of resistance to pathogens, and the like. These results can be achieved by providing the expression of heterologous products, the increased expression of endogenous products in plants, or the suppressed expression of endogenous products in plants.

General categories of polynucleotides of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in maintenance, such as heat shock proteins. More specific categories of transgenes include, for example, sequences encoding traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, oil loading, starch, carbohydrate, phytate, proteins, nutrients, metabolism. , digestible capacity, bone size, sucrose loading, and commercial products. Traits such as oil, starch, and protein content can be genetically altered. Modifications include increasing oleic acid content, saturated and unsaturated oils, increasing lysine and sulfur levels, providing essential amino acids, and also modifying starch. Modifications to the hordothionin protein to alter amino acid levels are described in US Patents 5,703,049, 5,885,801, 5,885,802, 5,990,389. Other examples are a lysine and / or sulfur rich seed protein encoded by 2S soy albumin described in US Patent 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson et al. (1987) Eur J Biochem 165: 99-106.

Derivatives of the coding sequences can be prepared to increase the level of preselected amino acids in the encoded polypeptide. For example, polynucleotides encoding barley high lysine (BHL) polypeptide are derived from barley chymotrypsin inhibitor (WO 98/20133). Other proteins include methionine-rich proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Illinois), p. 497-502); corn (Pedersen et al. (1986) J Biol Chem 261: 6279; Kirihara et al. (1988) Gene 71: 3); and rice (Musumura et al. (1989) Plant Mol Biol 12: 123).

Insect resistance polynucleotides can encode resistance to pests such as rootworm, cutter worm, European corn borer, and the like. Such polynucleotides include, for example, Bacillus thuringiensis toxic protein genes (US Patents 5,366,892; 5,747,450, 5,737,514; 5,723,756;

IS 2 390 132 T3

5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.

Polynucleotides encoding disease resistance traits include detoxification genes, such as against fumonisin (US Patent 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089 ); and the like.

Herbicide resistance traits can include genes encoding herbicide resistance that act by inhibiting the action of acetolactate synthase (ALS), particularly sulfonylurea-type herbicides such as chlorosulfuron (for example, the S4 and / or Hra mutations in ALS ); genes encoding herbicide resistance that act by inhibiting the action of glutamine synthase, such as phosphinothricin or coarse (eg, the bar gene); glyphosate (for example, the EPSPS gene or the GAT gene; see, for example, patent publications US20040082770 and WO 03/092360) or other known genes. Antibiotic resistance can also be provided, for example, by the nptll gene encoding resistance to the antibiotics kanamycin and geneticin.

Sterility genes can also be encoded on an expression cassette and provide an alternative to physical stripping of the male inflorescence. Examples of genes used in such a way include male tissue preferred genes and genes with male sterility phenotypes such as QM, described in US Patent 5,683,210. Other genes include kinases and those that encode compounds toxic to male or female gametophytic development.

Commercial traits can also be encoded in a gene or genes that could, for example, increase starch content for ethanol production, or provide protein expression. Another commercial use of transformed plants is the production of polymers and bioplastics as described in US Patent 5,602,321. Genes such as β-ketothiolase, PHBase (polyhydroxybutyrate synthase), and acetoacetyl-CoA reductase (see, Schubert et al. (1988) J Bacteriol 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs).

Reducing the activity of specific genes (also known as gene silencing, or gene suppression) is desirable for several aspects of genetic engineering in plants. Many gene silencing techniques are well known including, but not limited to, antisense technology (see, for example, Sheehy et al. (1988) Proc Natl Acad Sci USA 85: 8805-8809; and US Patents 5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g. Taylor (1997) Plant Cell 9: 1245; Jorgensen (1990) Trends Biotech 8: 340-344; Flavell (1994) Proc Natl Acad Sci USA 91: 3490-3496; Finnegan et al. (1994 ) Bio / Technology 12: 883-888; and Neuhuber et al. (1994) Mol Gen Genet 244: 230-241); RNA interference (Napoli et al. (1990) Plant Cell 2: 279-289; US Patent 5,034,323; Sharp (1999) Genes Dev 13: 139-141; Zamore et al. (2000) Cell 101: 25-33; Javier (2003) Nature 425: 257-263; and, Montgomery et al. (1998) Proc Natl Acad Sci USA 95: 15502-15507), virus-induced gene silencing (Burton, et al. (2000) Plant Cell 12: 691-705; and Baulcombe (1999) Curr Op Plant Bio 2: 109- 113); target RNA specific ribozymes (Haseloff et al. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000) Nature 407: 319-320; WO 99/53050; WO 02/00904; and WO 98/53083); ribozymes (Steinecke et al. (1992) EMBO J 11: 1525; US Patent 4,987,071; and, Perriman et al. (1993) Antisense Res Dev 3: 253); oligonucleotide-mediated targeted modification (eg WO 03/076574 and WO 99/25853); Zn finger-driven molecules (eg WO 01/52620; WO 03/048345; and WO 00/42219); and other known methods or combinations of the above methods.

Polynucleotides can be provided in a DNA construct. Furthermore, in specific examples, the recombination sites and / or the polynucleotide encoding an appropriate recombinase are also contained in the DNA construct. The cassette can include 5 'and 3' regulatory sequences operably linked to the polynucleotide of interest. Alternatively, the DNA construct flanked by the appropriate recombination site may lack the 5 'and / or 3' regulatory elements. In this case, the DNA construct is designed such that in the presence of the appropriate recombinase a recombination event at the target site causes the 5 'and / or 3' regulatory regions to be functionally linked to the sequences of the DNA construction. Intermediate sequences between functionally linked elements may be present and do not alter functional bonding. The cassette may additionally contain at least one additional gene to be introduced into the body. Alternatively, the additional gene (s) can be provided in multiple DNA constructs. Said DNA construct can be provided with a plurality of restriction sites or recombination sites for the insertion of the sequence of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selection and / or screening marker genes.

In some examples, the DNA Construct may include in the 5 'to 3' direction of transcription, a transcription and translation initiation region, a polynucleotide of interest, and a transcription and translation termination region functional in the body of interest. In other examples, the DNA construct comprises a polynucleotide of interest 3 'to a recombination site. In this example, the target site may comprise a promoter 5 'to the corresponding recombination site whereby, after recombination, the nucleotide sequence of interest is operably linked to the promoter sequence. The various recombination sites provided herein can be positioned anywhere in the construction of

IS 2 390 132 T3

DNA, including the 5 'UTR, 3' UTR, regulatory regions, introns, and / or coding sequence.

The transcriptional start region, the promoter, can be native, analogous, foreign, or heterologous to the host organism or polynucleotide of interest. Furthermore, the promoter can be the natural sequence or alternatively a synthetic sequence. Such constructs can change the expression levels of the polynucleotide of interest in the body. The termination region may be native or heterologous to the transcriptional initiation region, it may be native or heterologous to the polynucleotide of interest operably linked, or it may be native or heterologous to the host organism. Convenient termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol Gen Genet 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sarrfacon et al. (1991) Genes Dev 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res 17: 7891-7903; and Joshiet al. (1987) Nucleic Acids Res 15: 9627-9639. The nucleotide sequence of interest can also be native or analogous or foreign or heterologous to the host organism.

Where appropriate, the codon usage in the nucleotide sequence of interest or the recombinase can be modified for expression in the transformed organism. For example, genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol 92: 1-11 for a discussion of host preferred codon usage. Methods for synthesizing preferred plant genes are available. See, for example, US Patents 5,380,831, and 5,436,391, WO 99/25841, and Murray et al. (1989) Nucleic Acids Res 17: 477-498.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include the removal of sequences encoding spurious polyadenylation signals, exon-intron splicing and splicing signals, transposon-like repeats, and other such well-characterized sequences that can be detrimental to gene expression. The GC content of the sequence can be adjusted to average levels for a given cell host; calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted secondary hairpin mRNA structures.

The DNA construct may additionally contain 5 'leader sequences. Said leader sequences can act to enhance translation. Translation leaders are known and include leaders from picornavirus, eg, EMCV leader (encephalomyocarditis 5 'noncoding region) (Elroy-Stein et al. (1989) Proc Natl Acad Sci USA 86: 6126-6130); Potyvirus leaders, eg TEV (Tobacco Corrosion Virus) leader (Gallie et al. (1995) Gene 165: 233-238), MDMV (corn dwarf mosaic virus) leader (Allison et al. (1986) Virology 154: 9-20; and Kong et al. (1988) Arch Virol 143: 1791 -1799), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader of alfalfa mosaic virus coat protein mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus (TMV) leader (Gallie et al. (1989) in Molecular Biology of rNa, ed. Cech (Liss, New York), p. 237-256); and leader of the corn chlorotic mottle virus (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also, DellaCioppa et al. (1987) Plant Physiol 84: 965-968. Other methods or sequences known to enhance translation, eg, introns, and the like can also be used.

In preparing the DNA construct, the various DNA fragments can be manipulated to place the sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, hybridization, re-substitutions, transitions and / or transversions may be involved.

Generally, the DNA construct will comprise a selection marker gene for selection of transformed cells. Selection marker genes are used for the selection of transformed cells or tissues and have been discussed in detail elsewhere in this document, as well as exemplary promoters of interest.

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

Experimental

Reference example 1. Generation of libraries comprising modified FRT recombination sites.

Two complementary degenerate oligos containing FRT sequences with the 6 randomly mutagenized central spacer positions were synthesized in Synthetic Genetics (San Diego, CA): Oligo 1: 5'gccagcatgcaagcttgaattc-cgaagttcctatactNNNNNNagaataggaacttcgagatctggatggatccgcggaac; and Oligo 2: 5'-cgttccgcggatccagatctcgaagttcctattctNNNNNNagtataggaacttcggaattcaagcttgcatgctggc-3 '(SEQ ID No. 53).

The spacer region is 8 bp. In this experiment, the 6 bp central region was addressed for modification, for

Therefore, the other two nucleotides remain unchanged. An oligo 1 pmol and an oligo 2 pmol were hybridized by heat denaturation at 95 ° C for 2 minutes followed by gradual cooling to room temperature. The hybridized oligos were digested with EcoRI and BamHI and ligated into the EcoRI / BamHI sites of a vector derived from pSportI for which 3 additional bases created a Hpal restriction site (BRL Life Technologies, Gaithersburg, MD) and the vector PHP13273 that contained a spectinomycin resistance gene to create two modified FRT plasmid libraries. A 10: 1 and 4: 1 molar ratio of oligo hybridized to pSport and PHP13273 was used for the respective ligation reactions. Under these ligation conditions, 10 out of 10 randomly picked colonies were found to contain a monomeric insert of a modified FRT site. The modified FRT library referred to as library A is in the pSport vector and carries the antibiotic resistant marker ampicillin. The modified FRT library, referred to as library B, is in the vector PHP13273 and carries the antibiotic resistance marker spectinomycin. A total number of 15,904 colonies were picked to create FRT library A and 19,600 colonies were picked to create FRT library B. This represented 4x coverage of the 6 center positions (4⁶= 4096) on the FRT site. Plasmid DNA was isolated from these two libraries and used for library scale screening.

Reference Example 2. Library-scale screening to identify recombinogenic modified FRT recombination sites.

An equal molar amount of DNA from each of the modified FRT A and B libraries was mixed in a tube containing reaction buffer for FLP-mediated in vitro recombination. A typical 20 ml recombination reaction comprises 25 mM Tris Cl pH 7.5, 10 mM MgCl2, 5 mM DTT, 50 fmol DNA from library A, 50 fmol DNA from library B, and 2 ml FLP (0.07 mg / ml final). The reaction is carried out at 30 ° C, and aliquots are collected at various time points. At each point in time, 2 ml aliquots are collected and the reaction is stopped by boiling for 1 min. with gradual cooling to room temperature. Typically, samples are collected at 0, 2, 5, 10, 30, 60, and 90 minutes and could be used to evaluate fast versus slow reactive FRT sites. If only a specific moment was taken, the 90 minute point was used.

The reaction samples were transferred into E. coli DH5a cells according to standard procedures. Equal aliquots of each transformation mix were spread onto a plate each containing ampicillin only, spectinomycin only, or containing both ampicillin and spectinomycin. Successfully recombined co-integrating DNA will carry both selection markers and therefore, after transfer into E. coli, will confer resistance to both ampicillin and spectinomycin.

Those colonies with resistance to both antibiotics were picked and plasmid DNA was prepared using the Montage 96-well HTP plasmid DNA preparation kit (Millipore, Billerica, MA USA). Candidate FRT sites were obtained by PCR using primers flanking recombined FRT sites in the cointegrated DNA. The PCR primers used were the forward primer: 5'-gcacatacaaatggacgaacgga-3 (SEQ ID No. 54) and the reverse primer: 5'-cctcttcgctattacgccagct-3 '(SEQ ID No. 55). The PCR conditions were as follows: One cycle: 95 ° C, 1 min .; 20 cycles: 95 ° C, 30 seconds; 61 ° C, 2 min .; one cycle: 67 ° C, 3 min .; Maintenance: 4 ° C. The sequence of the amplified candidate FRT sites was determined by cycle sequencing (essentially as described in Slatko et al. (1993) DNA Sequencing. In, Current Protocols in Molecular Biology, (ed. By Ausubel et al.) Ch. 7 , pp. 7.0.1-7.6.13, New York: John Wiley & Sons).

Example 3. Methods for testing the efficiency of cleavage of recombinogenic modified FRT recombination sites.

To test the efficiency of recombinase-mediated cleavage of a candidate FRT site, cleavage vectors were prepared in which two copies of a candidate recombinogenic modified FRT site were cloned in direct orientation flanking the maize ubiquitin promoter sequence in pSport (BRL Life Technologies, Gaithersburg, MD). A cleavage reaction was performed under the following conditions: 3 ml of miniprep cleavage vector DNA (2 mg / ml), 1 ml of 10x buffer (250 mM Tris Cl at pH 7.5, 100 mM MgCl2, 50 mM DTT ), 5 ml of ddH2O, and 1 ml of FLP (0.72 mg / ml). The reaction mixture was incubated at 30 ° C for 30 min, boiled for 2 min, cooled to room temperature, digested with EcoRV and XhoI, and then subjected to agarose gel electrophoresis.

EcoRV generates a single cut in the pSport vector structure while XhoI generates a single cut in the maize ubiquitin promoter sequence. Double digestion of the non-recombined cleavage vector produces two 4332 bp and 769 bp fragments. Double digestion of the product vector after cleavage has taken place produces an additional 952 bp fragment. DNA fragments were quantified using Quantity One software from Bio-Rad Laboratories. As cleavage occurs, an increased amount of the 952 bp fragment is produced and less of the 769 bp fragment is produced. Thus, the ratio of the 952 bp fragment to the 769 bp fragment measures the absolute cleavage efficiency. In this experiment, the relative recombination cleavage efficiency (% cleavage efficiency) of one FRT site is calculated as the cleavage efficiency in the presence of native yeast FLP from a first modified FRT site with a second modified FRT site divided by the efficiency of cleavage of a pair of wild-type FRT sites (SEQ ID NO: 39) X 100%.

IS 2 390 132 T3

Various modified FRT recombination sites identified in the methods of Example 2 were tested for their ability to retain biological activity. Table 1 sets forth various functional modified FRT recombination sites and their relative recombination efficiency determined as outlined above. Data for FRT sites other than FRT1 and FRT12 are included for comparative purposes only.

Table 1

<td>FRT sites</td><td>SEQ ID No. for minimal modified FRT site</td><td>Sequence spacer</td><td>SEQ ID No. for modified spacer sequence</td><td>Cleavage efficiency (%)</td>

<td>FRT1</td><td> 39</td><td>TTTCTAGA</td><td> 43</td><td> 100</td>

<td>FRT12</td><td> 21</td><td>TCTATGTA</td><td> 1</td><td> 102</td>

<td>FRT57</td><td> 22</td><td>TTTTCTAA</td><td> 2</td><td> 82</td>

<td>FRT85</td><td> 23</td><td>TTTCTTGA</td><td> 3</td><td> 116</td>

<td>FRT87</td><td> 24</td><td>TTTCTGGA</td><td> 4</td><td> 93</td>

<td>FRT53</td><td> 25</td><td>TGTAAAAA</td><td> 5</td><td> 64</td>

<td>FRT62</td><td> 26</td><td>TTTAGGTA</td><td> 6</td><td> 72</td>

<td>FRT78</td><td> 27</td><td>TGAAAAGA</td><td> 7</td><td> 60</td>

<td>FRT34</td><td> 28</td><td>TGTAATGA</td><td> 8</td><td> 34</td>

<td>FRT70</td><td> 29</td><td>TATACAAA</td><td> 9</td><td> 25</td>

<td>FRT76</td><td> 30</td><td>TTCCATAA</td><td> 10</td><td> 30</td>

<td>FRT89</td><td> 31</td><td>TCTCTAGA</td><td> 11</td><td> 39</td>

<td>FRT43</td><td> 32</td><td>TTCCGAGA</td><td> 12</td><td> 14</td>

<td>FRT45</td><td> 33</td><td>TCTCTTGA</td><td> 13</td><td> 5</td>

<td>FRT55</td><td> 34</td><td>TCCACAGA</td><td> 14</td><td> 7</td>

<td>FRT65</td><td> 35</td><td>TGATTGGA</td><td> 15</td><td> 18</td>

<td>FRT69</td><td> 36</td><td>TTTTGTGA</td><td> 16</td><td> 9</td>

<td>FRT74</td><td> 37</td><td>TGAGAGAA</td><td> 17</td><td> 5</td>

<td>FRT86</td><td> 38</td><td>TTTCTCGA</td><td> 18</td><td> 12</td>

<td>FRT5</td><td> 40</td><td>CTTTTGAA</td><td> 44</td><td> 15</td>

<td colspan="5">The spacer sequences were flanked by the wild-type 13 base pair symmetric element depicted in Figure 1.</td>

Example 4. Methods for testing the efficiency of co-integration of recombinogenic modified FRT recombination sites.

The experiment was performed as described in Example 2. Briefly, FRT1, 5, and 6 (SEQ ID NO: 39, 40, and 41) were individually cloned into EcoRI / BamHl sites of PHP13273 and the modified vector pSportI. 50 fmol of FRT1 DNA in PHP13273 (Spec ') was mixed with 50 fmol of FRT1 DNA in modified pSportI (Ap') in 20 µl of reaction buffer containing 25 mM Tris Cl at pH 7.5, 10 mM MgCL , 5 mM DTT, and 2 µl FLP (0.07 µg / µl final). At each point in time, 2 µl aliquots were taken and the reaction was stopped by boiling for 1 min. with gradual cooling to room temperature. The reaction samples were transferred into E. coli DH5a cells according to standard procedures. Equal aliquots of each transformation mix were spread onto a plate, each containing ampicillin only, spectinomycin only, or containing both ampicillin and spectinomycin. Successfully recombined co-integrated DNA via FRT1 sites will carry both selection markers and therefore, after transfer into E. coli, will confer resistance to both ampicillin and spectinomycin. Those colonies with resistance to both antibiotics were picked and the co-integrated plasmid DNA was prepared for further analysis. Clones that are resistant to both antibiotics but do not harbor co-integrated plasmid DNA were subtracted in the calculation of the co-integration frequency.

The frequency of co-integration of FRT1 was determined by calculating the percentage of colonies harboring co-integrated plasmid DNA among the colonies resistant to an antibiotic drug. Also, in vitro integration of FRT5 or FRT6 was performed and the frequency of co-integration of FRT5 or FRT6 was determined accordingly. The results are shown in Table 2. The data for FRT5 and FRT6 are included for comparative purposes only.

Table 2. Percentage of co-members recovered from FLP-mediated recombination in vitro (%)

<td>Time (h)</td><td> 0</td><td> 0,5</td><td> 1,0</td><td> 1,5</td><td> 2,0</td>

<td>FRT1 + FRT1</td><td> 0,01</td><td> 0,32</td><td> 0,70</td><td> 0,98</td><td> 1,03</td>

<td>FRT5 + FRT5</td><td> 0,00</td><td> 0,04</td><td> 0,04</td><td> 0,08</td><td> 0,09</td>

<td>FRT6 + FRT6</td><td> 0,02</td><td> 0,20</td><td> 0,18</td><td> 0,28</td><td> 0,27</td>

In vitro FLP-mediated recombination was performed as described above. When DNA containing different FRT sites is mixed in the reaction, such as in the library-scale screening previously described, intermolecular recombination between two corresponding FRT sites is further reduced. In

ES 2 390 132 T3 reactions containing FRT1 sites only, FRT5 sites only, or FRT6 sites only, recombination between FRT1 sites is approximately 10 times more efficient than between FRT5 sites and approximately 4 times more efficient than between FRT6 sites (Table 2) .

In this example, plasmid DNA containing three different FRT sites (FRT1, FRT5, and FRT6) was pooled, each residing in modified pSportI carrying the selection marker Ap^r and PHP13273 that carries Spec^r, in the reaction. Between FRT1, FRT5, and FRT6, two different FRT sites do not recombine with each other. In the reaction having equimolar amounts of DNA containing FRT1, FRT5, and FRT6 FRT sites, the recombination efficiency between any two corresponding FRT sites is reduced. The results are shown in Table 3. The data for FRT5 and FRT6 are included for comparative purposes only. The frequency of combined co-integration between two FRT1 sites, two FRT5 sites, and two FRT6 sites was 0.09% after 90 minutes, approximately 10 times less than that of the reaction having the FRT1 site only. Most of the cointegrants are recombinant by the most efficient FRT sites, FRT1, in the reaction as indicated by the fact that the 10 randomly picked cointegrants were recombination products of FRT1 sites. In the reaction having a lower molar amount of DNA containing the FRT1 site (the molar ratio between FRT1, FRT5, and FRT6 is: 0.04: 1.00: 1.00), the overall recombination was further reduced. Furthermore, none of the 10 co-

<td colspan="3">Randomly chopped integrated was product of recombination of FRT1 sites. Table 3.</td><td></td>

<td>FRT sites *</td><td>Co-integrated (%)</td><td colspan="2">Co-integrated FRT1 / co-integrated total analyzed</td>

<td>FRT1 (Ap¹, 50 fmol) + FRT1 (Spec ', 50 fmol)</td><td> 0,98</td><td></td><td> 10/10</td>

<td>FRT5 (Ap^r, 50 fmol) + FRT5 (Spec^r, 50 fmol)</td><td> 0,08</td><td></td><td>NA</td>

<td>FRT6 (Ap^r, 50 fmol) + FRT6 (Spec^r, 50 fmol) FRT1 (Ap^r, 16 fmol) + FRT1 (Spec^r, 16 fmol) +</td><td> 0,28</td><td></td><td>NA</td>

<td>FRT5 (Ap^r, 16 fmol) + FRT5 (Spec^r, 16 fmol) + FRT6 (Ap^r, 16 fmol) + FRT6 (Spec^r, 16 fmol) FRT1 (Ap^r, 0.8 fmol) + FRT1 (Spec^r, 0.8 fmol)</td><td> 0,09</td><td></td><td> 10/10</td>

<td>FRT5 (Ap^r, 20 fmol) + FRT5 (Spec^r, 20 fmol) + FRT6 (Ap^r, 20 fmol) + FRT6 (Spec^r, 20 fmol)</td><td> 0,07</td><td></td><td> 0/10</td>

* The selection marker and the molar amount of DNA used in the reaction are included in parentheses.

Example 5: Transformation into plants

A. Transformation by particle bombardment and regeneration of corn callus

Immature corn embryos from greenhouse or field grown High type II (HiII) donor plants are bombarded with an isolated polynucleotide comprising a recombination site, transfer cassette, target site, and / or recombinase provided herein. If the polynucleotide does not include a selection marker, another polynucleotide containing a selection marker gene can be co-precipitated on the particles used for bombardment. For example, a plasmid containing the PAT gene (Wohlleben et al. (1988) Gene 70: 25-37) that confers resistance to the herbicide Bialafos can be used. The transformation is carried out as follows.

The pins are surface sterilized in 50% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed twice with sterile water. Immature embryos are excised and positioned with the embryonic axis down (scutellum up), 25 embryos per plate. These are grown on 560L agar medium 4 days before bombardment in the dark. 560L medium is an N6-based medium that contains Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. On the day of the bombardment, the embryos are transferred to 560Y medium for 4 hours and disposed within the 2.5 cm target zone. The 560Y medium is a highly osmostic medium (560L with high sucrose concentration).

A plasmid vector is constructed comprising a polynucleotide of interest operably linked to the selected promoter. This plasmid DNA, plus plasmid DNA containing a selection marker pAt if required, is precipitated into 1.0 mm (average diameter) gold granules using a CaCl2 precipitation procedure as follows: Prepare 100 ml of particles gold (0.6 mg) in water, 20 ml (2 mg) of DNA in TrisEDTA buffer (1 mg total), 100 ml of 2.5 M CaCl2, 40 ml of 0.1 M spermidine.

Each reagent is added sequentially to the gold particle suspension. The final mix is ​​sonicated briefly. After the precipitation period, the tubes are briefly centrifuged, the liquid is removed, washed with 500 ml of 100% ethanol, and centrifuged again for 30 seconds. Again the liquid is removed, and 60 ml of 100% ethanol is added to the final gold particle pellet. For particle gun bombardment, the gold / DNA particles are briefly sonicated and 5 ml is spotted onto the center of each macrocarrier and allowed to dry approximately 2 minutes prior to bombardment.

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The sample plates are bombarded at a distance of 8 cm from the stop screen to the tissue, using a DuPont biolistic helium particle gun. All samples are single shot at 4.48 MPa (650 PSI), with a total of ten aliquots taken from each prepared DNA / particle tube.

Four to 12 hours after bombardment, the embryos are moved to 560P (a low osmotic callus initiation medium similar to 560L but with lower silver nitrate content), for 3-7 days, then transferred to selection medium 560R, a 560P-like N6-based medium containing 3 mg / liter of Bialafos, and subcultured every 2 weeks. After approximately 10 weeks of selection, the callus clones are sampled for PCR and / or the activity of the polynucleotide of interest. Positive lines are transferred to 288J medium, an MS-based medium with lower levels of sucrose and hormones, to initiate plant regeneration. After somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to germination medium and transferred to the illuminated culture room. Approximately 7-10 days later, the developing seedlings are transferred to medium in tubes for 7-10 days until the seedlings are well established. The plants are then transferred to drawer inserts (equivalent to 6.35 cm (2.5) pots) containing compost soil and grown for 1 week in a culture chamber, then an additional 1-2 weeks are grown in the greenhouse, then transferred to Classic ™ 600 pots (6.06 L (1.6 gallons)) and grown to maturity. Plants are monitored for expression of the polynucleotide of interest.

B. Agrobacterium-mediated transformation and regeneration of corn callus

For Agrobacterium-mediated transformation of corn, a polynucleotide comprising a recombination site, transfer cassette, target site, and / or recombinase provided herein is used with the method of Zhao (US Patent 5,981,840).

Briefly, immature embryos are isolated from corn and the embryos are contacted with an Agrobacterium suspension containing a polynucleotide of interest, where the bacteria are capable of transferring the nucleotide sequence of interest to at least one cell of at least one of immature embryos (stage 1: the stage of infection). At this stage, immature embryos are immersed in an Agrobacterium suspension for initiation of inoculation. The embryos are co-cultured for a time with Agrobacterium (stage 2: the co-cultivation stage). After this co-cultivation period, a further resting stage can be carried out (stage 3: resting stage). Immature embryos are cultured in solid medium with antibiotic, but without selection agent, for the elimination of Agrobacterium and for resting anaphase for infected cells. The inoculated embryos are then cultured in medium containing a selective agent and the growing transformed callus is recovered (step 4: the selection step). Immature embryos are cultured in solid medium with a selective agent causing the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and the callus grown on selective medium are grown on solid medium to regenerate the plants.

C. Dicot transformation

A polynucleotide comprising a recombination site, transfer cassette, target site, and / or recombinase provided herein can be introduced into embryogenic suspension cultures of soybean by particle bombardment using essentially the methods described in Parrott, et al. (1989) Plant Cell Rep. 7: 615617. This method, with modifications, is described below.

The seeds are removed from the pods when the cotyledons are between 3 and 5 mm in length. The seeds are sterilized in a bleach solution (0.5%) for 15 minutes, after which time the seeds are rinsed with sterile distilled water. Immature cotyledons are excised by first cutting off the part of the seed that contains the embryonic axis. The cotyledons are then removed from the seed coat by gently pushing the distal end of the seed with the blunt end of the scalpel blade. The cotyledons are then placed in Petri dishes (flat up) with SB1 starter medium (MS salts, vitamins B5, 20 mg / L 2,4-D, 31.5 g / L sucrose, 8 g / L TC agar, pH 5.8). The Petri dishes are incubated in light (16 hrs; 75-80 mE) at 26 ° C. After 4 weeks of incubation, the cotyledons are transferred to fresh SB1 medium. After an additional two weeks, globular phase somatic embryos showing proliferative areas are excised and transferred to FN Lite liquid medium (Samoilov, et al. (1998) In Vitro Cell Dev. Biol.- Plant 34: 8-13) . Approximately 10 to 12 small groups of somatic embryos are placed in 250 ml flasks containing 35 ml of SB172 medium. Soybean embryogenic suspension cultures are kept in 35 ml of liquid medium on a rotary shaker, 150 rpm, at 26 ° C with fluorescent lights (20 mE) on a 16: 8 hour day / night program. The cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue in 35 ml of liquid medium.

The soybean embryogenic suspension cultures are then transformed using particle gun bombardment (Klein et al. (1987) Nature 327: 70; US Patent No. 4,945,050). A BioRad Biolistics PDS1000 / HE instrument can be used for these transformations. A selection marker gene, which is used to facilitate transformation of soybeans, is a chimeric gene composed of the 35S promoter of the cauliflower mosaic virus (Odell et al. (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25: 179-188) and the 3 'region of the nopaline gene plasmid T DNA synthase

IS 2 390 132 T3

Ti from Agrobacterium tumefaciens.

To 50 ml of a suspension of 1 mm gold particles at 60 mg / ml are added (in order): 5 ml of DNA (1 mg / ml), 20 ml of 0.1 M spermidine), and 50 ml of CaCl2 (2.5 M). The particle preparation is shaken for three minutes, centrifuged in a microfuge for 10 seconds, and the supernatant is removed. The DNA-coated particles are washed once in 400 ml of 70% ethanol and resuspended in 40 ml of anhydrous ethanol. The DNA / particle suspension is sonicated three times for one second each. Five ml of the DNA-coated gold particles are then loaded onto each macro carrier disk.

Approximately 300-400 mg of a two week suspension culture is placed in an empty 60x15 mm petri dish and residual liquid is removed from the tissue with a pipette. The rupture pressure of the membrane is set at 7.58 MPa (1100 psi) and the chamber is evacuated to a vacuum of 7.11 meters (28 inches) of mercury. The tissue is placed approximately 8 cm from the retention screen, and bombarded three times. After bombardment, the tissue is halved and placed back in 35 ml of FN Lite medium.

Five to seven days after bombardment, the liquid medium is exchanged with fresh medium. Eleven days after bombardment, the medium is exchanged with fresh medium containing 50 mg / ml hygromycin. This selective medium is refreshed weekly. Seven to eight weeks after bombardment, green transformed tissue will be seen growing from non-transformed necrotic embryogenic clumps. Isolated green tissue is removed and inoculated into individual flasks to generate new clonally propagated, transformed embryogenic suspension cultures. Each new line is treated as a separate transformation event. These suspensions are then cubed and maintained as groups of immature embryos, or tissue is regenerated into whole plants by maturation and germination of individual embryos.

D. Isolation of DNA from callus and leaf tissues

Putative transformation events can be screened for the presence of the transgene. Genomic DNA is extracted from callus or leaves using a modification of the CTAB (cetyltriethylammonium bromide, Sigma H5882) method described by Stacey and Isaac (1994 In Methods in Molecular Biology Vol. 28, pp. 9-15, Ed. PG Isaac , Humana Press, Totowa, NJ). Approximately 100-200 mg of frozen tissue are ground into a powder in liquid nitrogen and homogenized in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA, 0.1 M Tris-Cl pH 8, NaCl 1.4 M, 25 mM DTT) for 30 min. at 65 ° C. Homogenized samples are allowed to cool to room temperature for 15 min. before doing a single protein extraction with approximately 1 ml of 24: 1 v / v chloroform: octanol. The samples are centrifuged for 7 min. at 13,000 rpm and the top layer of supernatant is collected using wide-mouth pipet tips. DNA is precipitated from the supernatant by incubation in 95% ethanol on ice for 1 hr. The DNA strands are wound on a glass hook, washed in 75% ethanol containing 0.2M sodium acetate for 10 min, air dried for 5 min. and resuspended in TE buffer. Five ml of RNase A are added to the samples and they are incubated at 37 ° C for 1 hr. For the quantification of genomic DNA, gel electrophoresis is performed using a 0.8% agarose gel in 1x TBE buffer. One microliter of each of the samples is fractionated together with 200, 400, 600 and 800 ng ml^-1 of uncut DNA markers λ.

Reference Example 6. Comparison of the relative recombination efficiency of different FRT sequences in maize cells.

Two assays are provided that measure the relative activation rates of transgenes as a result of FLP-mediated cleavage, which brings a promoter and a transgene in functional proximity. The method can be used to characterize the recombination efficiency of corresponding and / or different recombination sites and thereby determine whether the sites are recombinogenic or non-recombinogenic with each other.

Two assays are discussed below: (A) titration of yellow fluorescent protein (YFP) activation in individual cells and (B) titration of luciferase activity.

A. Fluorescence assay

Three transgenic expression cassettes (summarized in Table 4) are introduced into an FRT test treatment or a control treatment.

Table 4.

<td>FRT test construction</td><td>Control construction</td>

<td>CPN60: FRTx: GUS: FRTx: YFP: 35s term</td><td>CPN60: FRTx: YFP: 35s term</td>

<td>Actin :: CFP :: us</td><td>Actin :: CFP :: us</td>

<td>Ubi :: FLP :: pinll</td><td>Ubi :: FLP :: pinll</td>

<td colspan="2">YFP = yellow fluorescent protein; CFP = cyan fluorescent protein; CPN60 = maize chaperonin 60 promoter (Close (1993) Master's Thesis, Iowa State University).</td>

IS 2 390 132 T3

In both the control treatment and the FRT test treatment, the three relevant (or appropriate) expression cassettes are mixed in equimolar proportions, and introduced into Hi-ll immature embryo scull cells using conventional particle delivery methods. . After two days, the amount of cyan and yellow fluorescent cells are counted using a Leica epifluorescent stereomicroscope. The amount of cyan fluorescent cells is used to normalize between treatments by providing a relative measure of how many cells received enough DNA to express the transgenes. To validate this assay system, FRT1 is used for the first experiment. As a control treatment, a mixture of the following three plasmids is used: Actin :: Cyan FP :: nos, CPN60: FRT1: YFP: 35s term, and Ubi :: FLP :: pinll. In the control treatment, when these three plasmids are co-introduced and the amounts of cyan and yellow cells are titrated two days later, the amounts of cyan and yellow cells in the population are expected to be approximately equivalent (1: 1).

In the FRT assay treatment, when FRT1 is used in the cleavage-activated cassette (CPN60: FRT1: GUS: FRT1: YFP: 35s term), approximately 90-95% of cells expressing cyan fluorescence are expected to also they express yellow fluorescence, that is, cleavage of the FRT1-flanked region is relatively efficient. Based on previous studies with FRT5, when FRT5 is used in the cleavage cassette, the frequency of cyan fluorescent cells also expressing yellow fluorescent protein is expected to drop to approximately 15% of that seen in FRT1 treatment.

The cleavage activated cassette can also be used to determine whether two different FRT recombination sites are recombinogenic or non-recombinogenic. To determine whether FRT1 and FRT5 are recombinogenic or non-recombinogenic with respect to each other, a cleavage-activated cassette is constructed comprising CPN60: FRT1: GUS: FRT5: YFP: 35s term. As summarized in Table 4, the three expression cassettes are mixed in equimolar ratios, and introduced into Hi-ll immature embryo scull cells using standard particle delivery methods. After two days, the amounts of cyan and yellow fluorescent cells are counted using a Leica epifluorescent stereomicroscope. The number of cyan fluorescent cells is used to normalize between treatments by providing a relative measure of how many cells received enough DNA to express the transgenes.

When the cleavage cassette comprises a FRT1 and FRT5 recombination site, the frequency of cyan fluorescent cells that also express the yellow fluorescent protein is expected to drop to approximately less than 1% of that observed when a cleavage cassette comprising two FRT1 recombination sites. The sites are therefore determined to be non-recombinogenic.

B. Assay based on activation of luciferase enzyme activity

The second test system again uses a mixture of plasmids in equimolar amounts, cobombarded in immature Hi-II embryo scull cells. For this assay, the three plasmids are shown in Table 5.

Table 5

<td>FRT trial treatment</td><td>Control</td>

<td>Actin :: FRTx: GUS: FRTx: FF-luciferase :: nos *</td><td>Actin :: FRTx: FF-luciferase :: nos *</td>

<td>Nos :: Renilla luciferase :: 35S term</td><td>Nos :: Renilla luciferase :: 35S term</td>

<td>Ubi :: FLP :: pinll</td><td>Ubi :: FLP :: pinll</td>

<td colspan="2">* FF = firefly luciferase; Renilla luciferase (Minko et al. (1999) Mol. Gen. Genet. 262: 421-425)</td>

Again, FRT1 is used to validate the assay system. In the control treatment, Actin: FRT1: FF-luciferase :: nos, Nos :: Renilla luciferase :: 35S term, and Ubi :: FLP :: pinll are introduced into skull cells and after 2 days, the tissue using methods and solutions provided in the Promega Double Luciferase Assay Kit (Promega, Madison, Wl 53711). Multiple scutelli are individually extracted, and the extracts are assayed sequentially for firefly luciferase activity and then Renilla using a Fluoroscan. With this mixture of constructs, the expressed firefly luciferase protein is expected to produce approximately 5000 relative units of light and the Renilla luciferase to produce approximately 15,000. When FRT1 is used in the cleavage activated cassette (Actin: FRT1: GUS: FRT1: firefly luci: 35s term), the firefly luciferase is expected to produce approximately 4500 light units (approximately 90% of the control treatment) . When FRT 5 is used in the cleavage cassette, the firefly luciferase activity is expected to drop to about 670 light units (~ 15% FRT1).

Reference example 7. Targeting the insertion of a polynucleotide of interest in maize.

A. Establishment of a target line.

For the evaluation of FRT sequences for site-specific integration, a target site is first created by integrating

ES 2 390 132 T3 stably a polynucleotide comprising a target site having two functional FRT recombination sites, where the recombination sites are different and non-recombinogenic with respect to each other. This initial transformation is achieved in Hi-II germplasm (or inbred lines) using standard transformation methods with Agrobacterium for maize (see Example 5B). For example, to compare the relative efficacy of FRT5 and FRT87 in the site-specific integration system, the following constructs are introduced separately into the Hi-II germplasm:

PHP20807:

RB- Ubi: Ubi-intron: FRT1: Yellow fluorescent protein :: pinll / Ubi: Ubi-intron:

GAT :: pinll / ln2-1 term: GUS: FRT5: Os-Actin-intron: Os-Actin Pro-LB PHP20705:

RB- Ubi: Ubi-intron: FRT1: Yellow fluorescent protein :: pinll / Ubi: Ubi-intron:

GAT :: pinll / ln2-1 term: GUS: FRT87: Os-Actin-intron: Os-Actin Pro-LB

Stable transformants are selected for yellow fluorescent calli growing on glyphosate-containing medium. The plants are regenerated and sent to the greenhouse. Foliar samples are taken for Southern analysis. Single copy transgenic plants are grown to maturity and crossed with wild type Hi-II (or inbred lines). These transgenic events now contain the FRT1-5 or FRT187 target site, and are ready for site-specific recombinase-mediated recombination.

B. Insertion of the transfer cassette using particle bombardment.

Immature embryos of the line having the target sites as evidenced by expression of yellow fluorescence are used for subsequent retransformation. During the retransformation process, the transfer cassettes are introduced using conventional particle bombardment methods (eg, see Example 5A). For offspring plants containing the integrated T DNA of PHP20705 (the FRT1-FRT87 target site), the following insert comprising the transfer cassette is used for retransformation using the particle gun:

PHP20915:

RB- CaMV35S Term / FRT1: bar :: pinll / Ubi: Ubi-intron: Renilla luciferase :: pinll / In2-1 term: Am-Cyan1:

FRT87 / CaMV35S Term -LB.

The immature embryos of the offspring containing the integrated T DNA of PHP20807 (the FRT1-FRT5 target site) are retransformed using the particle gun with the following plasmid:

PHP20954:

RB- CaMV35S Term / FRT1: bar :: pinll / Ubi: Ubi-intron: Renilla luciferase :: pinll / In2-1 term: Am-Cyan1:

FRT5 / CaMV35S Term -LB.

For the two plasmids that comprise the transfer cassette (PHP20915 and PHP20954), the bar and Cyan FP genes have no promoter. To reduce the likelihood that random integration will not cause false expression of any gene, the CaMV35S terminator has been placed upstream of the FRT1 site. Each of these plasmids is co-transformed into immature embryos for their respective target lines together with plasmid PHP5096 (Ubi: Ubi-intron :: FLPm :: pinII). Either PHP20915 or PHP20954 is mixed with the FLP-containing plasmid (PHP5096), using 100 ng of the FRT-containing plasmid and 10 ng of the FLP plasmid per bombardment.

To prepare the DNA for delivery, DNA solutions are added to 50 ml of a gold particle stock solution (0.1 mg / ml 0.6 micron gold particles). For example, 10 ml of a 0.1 mg / ml solution of PHP20915 or PHP20954, and 10 ml of a 0.01 mg / ml solution of PHP5096 are first added to 30 ml of water. To this DNA mixture, 50 ml of the gold stock solution is added and the mixture is briefly sonicated. Then 5 ml of TFX-50 (Promega Corp., 2800 Woods Hollow Road, Madison WI 53711) is added and the mixture is put on a rotary shaker at 100 rpm for 10 minutes. The mixture is centrifuged briefly to pellet the gold particles and remove the supernatant. After removing excess DNA / TFX solution, 120 ml of absolute EtOH are added, and 10 ml aliquots are dispensed onto the macrocarriers typically used with the Dupont PDS-1000 helium particle gun. The DNA-attached gold particles are allowed to dry on the carriers and the carriers are then used for conventional particle bombardment. After providing retransformation of the plasmid containing the transfer cassette plus the plasmid containing FLP, the immature embryos are placed in 560P medium for two weeks for recovery, and then moved to medium containing 3 mg / L bialaphos for the selection. Successful recombination at both the 5 '(FRT-1) and 3' (FRT87 or 5) recombination target sites will cause activation of both the bar gene and the cyan fluorescent protein gene when

ES 2 390 132 T3 these structural genes are brought into functional proximity to the Ubi or Actin promoters, respectively. Therefore, appropriate site-specific integration events will be selected based on the newly activated phenotypes. When these calli are large enough for sampling, genomic DNA is extracted from the tissue, and analyzed using PCR for the presence of products resulting from fragment amplification using primers that span the newly formed promoter-gene junctions. Finally, foliar samples of regenerated plants are taken for Southern analysis, to verify proper recombination for transfer of the donor cassette at the genomic target locus. Once satisfactory recombinant loci have been verified, the plants are grown to maturity and crossed or self-crossed.

C. Introduction of the crossover transfer cassette

Transfer cassettes can be provided for sexual reproduction. In this example, single copy target stable transgenic events containing a single copy of the originally supplied Agrobacterium T-DNA cassettes containing PHP20705 or PHP20807 are again used. However, in this method stable transgenic donor events are produced using either of the two T-DNA Agrobacterium vectors shown below.

1. The donor vector that complements PHP20705:

RB-Axig1 :: IEC1 :: pinll / Ubi Pro: Ubi-intron :: YFP :: pinll / -LB y

RB- ln2 :: FLP :: pinll-CaMV35S Term / FRT1: bar :: pinll / Ubi: Ubi-intron: Renilla luciferase :: pinll / ln2-1 term: AmCian1: FRT87 / CaMV35S Term -LB

2. The donor vector that complements PHP20807:

RB-Axig1 :: LEC1 :: pinll / Ubi Pro: Ubi-intron :: YFP :: pinll / -LB y

RB-ln2 :: FLP :: pinll-CaMV35S Term / FRT1: bar :: pinll / Ubi: Ubi-intron: Renilla luciferase :: pinll / In2-1 term: AmCian1: FRT5 / CaMV35S Term -LB

For the above two plasmids, the expression cassettes in the first T-DNA provide a means of selecting transgenic donor lines after Agrobacterium-mediated transformation. The second T DNA provides the components for crossover mediated cassette exchange. Note that for both constructs, the inducible FLP expression cassette is outside the FRT sites and thus it is not transferred to the target site after successful exchange.

Recombination events having the transfer cassette are selected by visual selection for regenerated, vigorously growing yellow fluorescent calli, grown to maturity and crossed to produce donor seeds bearing the transfer cassette. The seed of a target event containing the PHP20705 T DNA fragment as well as the seed of a donor event containing the T DNA of the donor plasmid # 1 above are planted and grown to maturity. After flowering, reciprocal crosses are made between the target and donor plants. The resulting seed is planted and selected for newly activated phenotypes indicating successful recombination at the two different FRT sites. In this case, activation of bialaphos resistance is indicative of appropriate recombination in FRT5 and activation of cyan fluorescence is indicative of appropriate recombination in FRT87. Similar crosses are made using target and blot lines generated with PHP20807 and donor plasmid # 2, respectively.

Example 8. Transformation of bacterial cells.

The new recombination sites provided herein can also be evaluated and used in bacterial cells, such as E. coli. Many commercially available competent cell lines and bacterial plasmids are well known and readily available. Isolated polynucleotides for transformation and transformation of bacterial cells can be done by any method known in the art. For example, methods of E. coli and other bacterial cell transformations, plasmid preparation, and the use of phages, for example, in Current Protocols in Molecular Biology (FM Ausubel et al., (eds.) (1994) a joint venture between Greene Publishing Associates, Inc . and John Wiley &amp; Sons, Inc.). For example, an effective electroporation protocol is summarized below (Tung and Chow, Current Protocols in Molecular Biology, Supplement 32, Fall 1995).

100 ml of LB medium are inoculated with 1 ml of E. coli culture overnight. They are incubated at 37 ° C with vigorous shaking until the culture reaches OD600 = 0.6. The culture is cooled on ice for 30 minutes, the cells are pelleted by centrifugation at 4,000 xg for 15 minutes at 4 ° C. The cell pellet is washed twice with 50 ml of ice cold 10% glycerol. After the final wash, the cell pellet is resuspended to a final volume of 0.2 ml in ice-cold GYT medium (10% v / v glycerol; 0.125% w / v yeast extract; 0.25% tritone w / v). They are electroporated in pre-cooled cuvettes using manufacturer's conditions, for example, 0.5 ng plasmid DNA / transformation using Gene Pulser equipment (BioRad) for 25 mF, 200 ohms, 2.5 kV. Immediately after electroporation, 1 ml of SOC medium is added and the cells are transferred to a tube.

ES 2 390 132 T3 of culture. They are incubated at 37 ° C for 1 hour. Aliquots of cells are seeded onto selective agar plates and incubated overnight at 37 ° C.

Example 9. Yeast transformation.

The new recombination sites provided herein can also be evaluated and used in yeast cells, from which the FLP recombinase and FRT sites were initially isolated. Many commercial and / or public strains of S. cerevisiae are available, as are the plasmids used to transform these cells. For example, cells are available from the American Type Culture Collection (ATCC, Manassas, VA) and includes the inventory from the Yeast Genetic Stock Center, which was moved to ATCC in 1998. Other yeast lines are also available, such as S. pombe and P. pastoris, and the like. For example, yeast transformation methods, plasmid preparation, and the like are detailed, for example, in Current Protocols in Molecular Biology (FM Ausubel et al., (Eds.) (1994) a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., see Unit 13 in particular). Transformation methods for yeast include spheroplast transformation, electroporation, and lithium acetate methods. A highly efficient, versatile transformation method for yeast is described by Gietz and Woods ((2002) Methods Enzymol. 350: 87-96) using lithium acetate, PEG 3500 and carrier DNA.

Example 10. Transformation of mammalian cells.

The new recombination sites provided herein can also be evaluated and used in mammalian cells, such as CHO, HeLa, BALB / c, fibroblasts, mouse embryonic stem cells, and the like. Many commercially available competent cell lines and plasmids are well known and readily available, for example from ATCC (Manassas, VA). Isolated polynucleotides for transformation and transformation of mammalian cells can be made by any method known in the art. For example, methods of transformation of mammalian and other eukaryotic cells, preparation of plasmids, and the use of viruses are detailed, for example, in Current Protocols in Molecular Biology (FM Ausubel et al. (Eds.) (1994) a company joint venture between Green Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., see Unit 9 in particular). For example, many methods are available, such as calcium phosphate transfection, electroporation, DEAE-dextran transfection, liposome-mediated transfection, microinjection as well as virus techniques.

Reference example 11. Methods for in vitro recombinational cloning.

In the following Examples A, B, and C, the two parental nucleic acid molecules (eg, plasmids) are called the insert donor and the vector donor. The insert donor contains a segment that will become attached to a new sequence contributed by the vector donor. The recombination event produces two daughter molecules: the first mentioned as the product (the new desired clone) and the second mentioned as the by-product.

In the following examples, two pairs of plasmids are constructed to perform the in vitro recombinational cloning method in two different ways. A pair of plasmids, Plasmid A and Plasmid B, with a FRT site and a lox site, are constructed for use with the Cre and FLP recombinase. The other pair of plasmids, Plasmid D and Plasmid E, are constructed to contain the FRT site (wild-type) for fLp, and a second mutant FRT site (SEQ ID No. 40), which differs from the wild-type FRT site. on 3 of the 30 total bases. In this example, each plasmid comprises a series of functional recombination sites where the recombination sites are different and non-recombinogenic with respect to each other.

Buffers:

Various known buffers can be used in the reactions. For restriction enzymes, it is advisable to use the buffers recommended by the manufacturer. Alternative buffers can easily be found in the literature or can be deduced by those skilled in the art. An exemplary buffer for lambda integrase comprises 50 mM Tris-HCl pH 7.5-7.8, 70 mM KCl, 5 mM spermidine , 0.5 mM EdTA, and 0.25 mg / ml bovine serum albumin, and optionally 10% glycerol. An exemplary buffer for P1 Cre recombinase comprises 50 mM TrisHCl, pH 7.5, 33 mM NaCl, 5 mM spermidine, and 0.5 mg / ml bovine serum albumin, and an exemplary buffer for FLP has been previously discussed in Example 2. An exemplary buffer for Cre and FLP recombinases comprises 50 mM Tris-HCl, pH 7.5, 70 mM NaCl, 2 mM MgCL, and 0.1 mg / ml BSA (Buchholz et al. (1996 ) Nucleic Acids Res. 24: 4256-4262). Buffer for other site-specific recombinases is known in the art or can be determined empirically by those skilled in the art, particularly in light of the buffers described above.

A. Recombinative cloning using FLP recombinase.

Two plasmids are constructed. The donor plasmid (plasmid A) comprises in the following order: a wild-type FRT site, a constitutive drug marker (chloramphenicol resistance), an origin of replication, a gene

ES 2 390 132 T3 constitutively expressed for the tet repressor protein (tetR), an FRT 5 site, and a conditional drug marker (kanamycin resistance whose expression is controlled by the operator / promoter of the transposon tetracycline resistance operon Tn10). E. coli cells containing plasmid A are resistant to chloramphenicol at 30 mg / ml, but sensitive to kanamycin at 100 mg / ml.

The insert donor plasmid (plasmid B) comprises in the following order: the wild-type FRT site, a different drug marker (ampicillin resistance), the FRT 5 site, an origin, and a multiple cloning site.

Approximately 75 ng of each of plasmids A and B are mixed in a total volume of 30 ml of FLP reaction buffer. Two 10 ml aliquots are transferred to new tubes. One tube receives FLP protein. Both tubes are incubated at 37 ° C for 30 minutes, then at 70 ° C for 10 minutes. Aliquots from each reaction are diluted and transformed into DH5a. After expression, aliquots are plated in 30 mg / ml chloramphenicol; 100 mg / ml ampicillin plus 200 mg / ml methicillin; or 100 mg / ml kanamycin.

Colonies that are chloramphenicol resistant, ampicillin resistant and kanamycin sensitive underwent the recombination reaction and comprise the newly generated product vector (plasmid C). Plasmid C comprises in the following order: wild-type FRT site, constitutive drug marker (chloramphenicol resistance), origin of replication, constitutively expressed gene for tet repressor protein (tetR), FRT site 5, and the ampicillin resistance marker.

To confirm the structure of the product vector (plasmid C), colonies that are chloramphenicol resistant, ampicillin resistant, and kanamycin sensitive are picked and inoculated into medium containing 100 mg / ml kanamycin. Minipreps are made and the miniprep DNAs are cut with the appropriate restriction enzymes and electrophoresed. Plasmid C can be identified based on the predicted size for the product plasmid and fragments resulting from restriction enzyme digestion.

B. Recombinational cloning using FLP recombinase and Cre recombinase.

The plasmids of this method are analogous to the above, except that plasmid D, the vector donor plasmid, contains a loxP site in place of the wild-type FRT site, and plasmid E, the insert donor plasmid, contains the site loxP instead of the wild-type FRT site.

Approximately 500 ng of plasmid E and plasmid D are ethanol precipitated and resuspended in 40 ml of Cre / FLP reaction buffer (described above). Reactions are incubated at 37 ° C for 30 minutes and then at 70 ° C for 10 minutes. TE buffer (90 ml, TE: 10 mM Tris HCl, pH 7.5, 1 mM EDTA) is added to each reaction, and 1 ml of each is transformed into E. coli DH5a. Reaction mixtures are plated in 100 mg / ml ampicillin plus 200 mg / ml methicillin; 30 mg / ml chloramphenicol or 100 mg / ml kanamycin.

Colonies that are chloramphenicol resistant, ampicillin resistant and kanamycin sensitive underwent the recombination reaction and comprise the newly generated product vector (plasmid F). Plasmid F comprises in the following order: wild-type loxP site, constitutive drug marker (chloramphenicol resistance), origin of replication, constitutively expressed gene for tet repressor protein (tetR), FRT site 5, and the ampicillin resistance marker.

To confirm the structure of the product vector (plasmid F), colonies that are chloramphenicol resistant, ampicillin resistant, and kanamycin sensitive are picked and inoculated into medium containing 900 mg / ml kanamycin. Minipreps are made and the miniprep DNAs are cut with the appropriate restriction enzymes and electrophoresed. Plasmid F can be identified based on the predicted size for the product plasmid and fragments resulting from restriction enzyme digestion.

C. In vitro recombinational cloning to subclone the chloramphenicol acetyl transferase gene into a vector for expression in eukaryotic cells.

An insert donor plasmid, plasmid G, is constructed comprising in the following order: a wild-type FRT site, an E. coli chloramphenicol acetyl transferase gene lacking a promoter, the FRT 5 site, an origin of replication , and a constitutive drug marker (ampicillin resistance).

A vector donor plasmid, plasmid H, is constructed comprising in the following order: kanamycin resistance gene, origin of replication, the eukaryotic promoter of cytomegalovirus, a wild-type FRT site, the constitutively expressed gene for the protein tet repressor (tetR), a chloramphenicol resistance gene, and the FRT 5 site. One microliter aliquots of each plasmid, typically about 50 ng of crude miniprep DNA, are combined into a 10 ml reaction containing FLP reaction buffer and FLP recombinase. After incubation at 30 ° C for 30 minutes and 75 ° C for 10 minutes, one microliter is transformed into the competent E. coli strain DH5a (Life Technologies, Inc.). Aliquots of transformations are spread onto agar plates containing 200 mg / ml kanamycin and incubated at 37 ° C overnight. A control reaction so