PL1907553T3

Modified frt recombination sites and methods of use

Abstract

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8 claims: 3 independent, 5 dependent

1
Patent claims Zastrzeżenia patentowe 1. 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. 1. Wyizolowany polinukleotyd zawierający sekwencję nukleotydową zawierającą przynajmniej pierwsze miejsce rekombinacji FRT obejmujące SEQ ID NO: 21 oraz drugie miejsce rekombinacji FRT obejmujące SEQ ID NO: 39.
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A plant comprising a cell as defined in claim 3. 4. Roślina zawierająca komórkę określoną w zastrz. 3.
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A method of oriented insertion of a desired polynucleotide into a target site, said method comprising:6. Sposób ukierunkowywanego wstawiania pożądanego polinukleotydu do docelowego miejsca, przy czym wspomniany sposób obejmuje: (a) dostarczenie komórki posiadającej stabilnie zintegrowane z jej genomem docelowe miejsce zawierające pierwsze miejsce rekombinacji obejmujące SEQ ID NO: 21 oraz drugie miejsce rekombinacji obejmujące SEQ ID NO: 39;(a) providing a cell having a target site stably integrated into its genome comprising a first recombination site comprising SEQ ID NO: 21 and a second recombination site comprising SEQ ID NO: 39;(b) dostarczenie kasety transferowej zawierającej pożądany polinukleotyd, przy czym wspomniany, pożądany polinukleotyd jest flankowany przez wspomniane pierwsze i drugie miejsce rekombinacji;oraz (c) dostarczenie rekombinazy FLP, przy czym wspomniana rekombinaza rozpoznaje i przeprowadza rekombinację w pierwszym i drugim miejscu rekombinacji a w docelowym miejscu wstawiany jest pożądany polinukleotyd. (b) providing a transfer cassette containing the desired polynucleotide, said desired polynucleotide being flanked by said first and second recombination sites;and (c) providing FLP recombinase, said recombinase recognizing and performing recombination at the first and second recombination sites, and the desired polynucleotide is inserted at the target site.

Independent claims3

922 paragraphs in 32 sections, as filed

[0001] The invention relates to site-specific recombination systems and methods of their use.

BACKGROUND OF THE INVENTION [0002] Accidental insertion of DNA introduced into the host cell genome may prove lethal if the insertion of foreign DNA causes a mutation within the native gene of significant importance. Furthermore, even if accidental insertion does not adversely affect the functioning of the host cell gene, the expression effect of the inserted foreign nucleotide sequence may be affected by the location effect induced by the genomic DNA surrounding this gene. In some cases, the nucleotide sequence is inserted at a place where the location effect is so strong that it suppresses the function or regulation of the introduced nucleotide sequence. In other cases, overproduction of this gene product has a detrimental effect on the cell. [0003] For example, in plants, the location effect may result in reduced crop, additional costs for further research, the emergence of new transgenic cases, and a slower time to obtain the product. For this reason, there is a need for efficient methods for directing the insertion of nucleotide sequences in the genome of various organisms, such as plants, at chromosomal positions that allow obtaining the desired functions for which the sequence of interest is responsible.

[0004] Schlake T. and Bode, J. (Biochem. (1994) 33: 12746-51) disclosed the use of mutated FLP (FRT) recognition sites to exchange expression cassettes at specific chromosomal loci.

Summary [0005] Methods and compositions have been disclosed using a population of randomly modified FRT recombination sites for the identification, isolation and / or characterization of modified FRT recombination sites. Recombinable modified

FRT recombination sites can be used in various methods of directed recombination of desired polynucleotides, including recombinant polynucleotide methods, evaluation of promoter activity, directly selected transformed organisms, minimization or elimination of expression resulting from random integration with the genome of the organism, such as plant, as well as removal of desired polynucleotides, combining multiple transfer cassettes, polynucleotide inversion or excision and identification and / or characterization of regulatory transcription regions.

[0006] The invention relates to 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.

[0007] The invention also relates to a cell containing the polynucleotide of the invention.

[0008] The invention also relates to a plant comprising a cell according to the invention.

[0009] The invention also relates to a method of directing the insertion of a desired polynucleotide at a target site, said method comprising:

(a) providing a cell having a target site stably integrated in its genome 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 containing the desired polynucleotide, said desired polynucleotide being flanked by said first and said second recombination site; and (c) providing FLP recombinase, wherein said recombinase recognizes and performs recombination at the first and second recombination sites and the desired polynucleotide is inserted at the target site.

DETAILED DESCRIPTION OF THE INVENTION [0010] The invention relates to an isolated polynucleotide comprising a nucleotide sequence comprising a first functional modified FRT recombination site comprising the nucleotide sequence listed in SEQ ID NO: 21 and a second FRT recombination site comprising SEQ ID NO: 39.

[0011] Also disclosed are organisms including, for example, prokaryotic organisms such as bacteria and eukaryotic organisms such as yeast, plants, plant cells and seeds containing said polynucleotides including modified FRT recombination sites . In specific examples, polynucleotides are stably integrated into the genome of the body.

[0012] A method of targeting insertion of a desired polynucleotide is disclosed.

The method comprises a target comprising the first and second functional recombination sites of the invention. A transfer cassette is provided containing the desired polynucleotide and two functional recombination sites, wherein the second functional recombination site is capable of recombination with the first functional recombination site, and the first and / or second recombination site comprises the modified FRT site disclosed herein. At least one recombinase is provided. Recombinase recognizes and performs recombination in the first and second recombination sites. The method can be carried out in vitro or in vivo. Functional recombination sites are stably integrated into the cell genome at the target site. In some examples, the desired polynucleotide and / or target may then be excised, inverted or otherwise modified, for example, by adding a second desired polynucleotide at the target site.

[0013] The minimal wild-type FRT recombination site has been characterized, it contains a series of domains, including the following 5'AGTTCCTATTCTCTAGAAAGTATAGGAACT-3 'nucleotide sequence (SEQ ID NO: 39). The FRT minimal recombination site domain contains a pair of 11 parobasic symmetrical elements that are FLP binding sites (nucleotides 1-11 and 20-30 of SEQ ID NO: 39); the 8 base pair length core or linker sequence, region (nucleotides 12-19 of SEQ ID NO: 39) and the polypyrimidine pathway (nucleotides 3-14 and nucleotides 16-29 of SEQ ID NO: 39). The modified FRT recombination site may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more changes that include substitutions, addition and / or excision in one or more of these domains.

[0014] A modified FRT recombination site was disclosed. The modified FRT recombination site is a nucleotide sequence that is similar but not identical to the minimum native FRT recombination site specified in SEQ ID NO: 39.

The modified FRT recombination site retains the biological activity of the wild type FRT recombination site and contains a functional recombination site that is recognized by FLP recombinase and is able to participate in the process

Recombination in which recombinase is involved. The modified FRT recombination site contains substitution of one or more nucleotides at one or more internal sites at the minimal native FRT recombination site.

The modified FRT recombination site contains SEQ ID NO: 21. Generally, the modified recombination sites will exhibit at least about 40%, 45%,

50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,

85%, 86% sequence identity to the minimum native recombination site along its entire length.

[0015] The modified FRT recombination site has nucleotide substitutions along the entire length of the minimum recombination site.

[0016] The modified FRT recombination site has changes in the 8-base domain of the linker sequence. The modified FRT12 linker domain is defined in SEQ ID NO: 1. The modified FRT site is functional.

The modified FRT recombination site contains the linker region defined in SEQ

ID NO: 1, and also has a symmetrical element of FLP binding sites that correspond to sites that occur at the minimum native recombination site

FRT. See, SEQ ID NOS: 19 and 20 showing the wild type sequence of the symmetric element. The modified FRT recombination site is specified in SEQ ID NO:

21.

[0017] The modified FRT site comprising SEQ ID NO: 21 is used in combination with the wild type FRT site containing SEQ ID NO: 39 in the composition or method of the invention.

[0018] As mentioned above, the modified recombination site is functional. A functional recombination site is a recombination site that recombines (is recombinogenic) with a recombination site in the presence of a suitable recombinase and, unless otherwise mentioned, the recombination site is functional and includes wild-type sites, modified sites, variants and fragments. Methods to determine whether a modified recombination site is recombinogenic are known. As used herein, a variant of a functional recombination site comprises a functional, modified site.

[0019] The recombination sites used in the method are different sites.

Alternate recombination sites or a set of distinct recombination sites are recombination sites that differ from each other by at least one nucleotide.

Recombination sites in a set of distinct recombination sites may be recombinogenic or non-recombinogenic relative to another site. The term recombinogenic refers to recombination sites capable of being recombined

PZ / 1656 / AR EP 1 907 553 B1 with another place. Unless otherwise indicated, recombinogenic recombination sites or a set of recombinogenic recombination sites include sites where the relative excision efficiency during recombination between sites is greater than 2%, 5%, 10%, 20%, 30%, 40%, 50% , 75%, 100% or higher. As defined herein, the relative recombinant excision efficiency is the excision efficiency in the presence of native recombinase of the first modified recombination site with the second modified recombination site divided by the cleavage efficiency of the pair of corresponding native X 100% recombination sites. For example, using modified places

FRT, relative recombinant cleavage efficiency is defined as cleavage efficiency in the presence of native FLPs (SEQ ID NO: 49) for the first modified FRT site with the second modified FRT site divided by the notch performance for a pair of native FRT sites (FRT1, SEQ ID NO: 39 ).

The term non-recombinogenic refers to recombination sites that, in the presence of the appropriate recombinase, do not recombine with the second site, or recombination between sites occurs at a minimal level. Unless otherwise mentioned, non-recombinant recombination sites or a set of recombinogenic recombination sites include those sites for which the relative recombinant excision efficiency between sites is less than 2%, 1.5%, 1%,

0.75%, 0.5%, 0.25%, 0.1%, 0.075, 0.005%, 0.001%.

[0020] A modified FRT recombination site containing SEQ ID NO: 21 is contained in a polynucleotide with a FRT site comprising SEQ ID NO: 39. In one example, the polynucleotide contains one or more expression units. The expression unit is a nucleotide sequence containing a DNA unit characterized by the content of a single transcriptional promoter. Alternatively, a polynucleotide containing a modified FRT recombination site need not contain a promoter and / or underlying regulatory sequences. In other examples, a polynucleotide containing a modified recombination site may be designed such that when incorporated into the genome, the sequences contained in the polynucleotide are operably linked to an active promoter. It is believed that the polynucleotide may have additional elements, including, but not limited to, desired nucleotide sequences, marker genes, recombination sites, termination regions, etc. As illustrated below, the polynucleotide may contain transfer cassettes, targets or any fragments thereof.

[0021] The isolated or purified polynucleotide or protein, or a biologically active fragment thereof, is substantially or substantially free of components that normally

PZ / 1656 / AR EP 1 907 553 B1 accompanies or interacts with a polynucleotide or protein present in its natural environment. An isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when obtained by recombination techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically.

Typically, an isolated polynucleotide does not contain sequences that naturally flank the 5 'and / or 3' ends of the polynucleotide in the genomic DNA of the organism from which the polynucleotide originates. For example, in various examples, an 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 a nucleotide sequence that naturally flanks the polynucleotide in the cell's genomic DNA, where the polynucleotide comes from A protein that is substantially free of cellular material includes protein preparations having less than about 30%, 20%, 10%, 5% or 1% dry weight of the contaminating protein. When a protein or biologically active fragment thereof is produced by recombinant technology, generally the culture medium is less than about 30%, 20%, 10%, 5% or 1% (on a dry weight basis) of chemical precursors or chemical components that are not the desired protein.

Polynucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. Polynucleotides also include all sequence forms including, but not limited to, single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, and the like.

[0022] In one example, an isolated polynucleotide is provided, wherein the polynucleotide comprises a modified FRT recombination site comprising SEQ

ID NO: 21 and a FRT site comprising SEQ ID NO: 39. In specific examples, the modified FRT recombination site is a polynucleotide sequence. In specific examples, a modified recombination site

FRT is heterologous to the polynucleotide.

[0023] Heterologous refers to a polypeptide or nucleotide sequence that is from different species, or if from the same species that is substantially modified relative to its native form in the composition and / or the genomic locus.

For example, a heterologous recombination site is a polynucleotide that does not occur in the native polynucleotide or does not occur in the same position in the native polynucleotide and / or is modified in its native form composition.

[0024] In other examples, an isolated polynucleotide is provided that comprises the nucleotide sequence set out in SEQ ID NO: 21 and the nucleotide sequence set out in SEQ ID NO: 39.

[0025] The modified FRT recombination site may be introduced into the appropriate organism. The introduction includes presenting to the body at least one molecule, composition, polynucleotide or polypeptide in such a way that the composition gains access to the interior of the cell. The methods do not depend on the particular route of introduction of the polynucleotide or polypeptide into the body, it is only important that the polynucleotide or polypeptide has access to the interior of at least one cell of the body.

[0026] Suitable organisms 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.

[0027] Methods for delivering or introducing compositions to various organisms are known and include, but are not limited to, stable transformation methods, transient transformation methods, viral methods, and sexual reproduction. Stable transformation means that the introduced polynucleotide is integrated into the genome of the body and is inherited by its offspring.

Transient transformation means that the introduced composition is only temporarily expressed or temporarily present in the body.

[0028] Protocols for introducing polynucleotides and polypeptides into plants may vary depending on the type of plant undergoing transformation, such as a monocotyledonous or dicotyledonous plant. Suitable methods for introducing polynucleotides and polypeptides into plant cells and then inserting them 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, transformation using Agrobacterium (Patents

United States of America 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)

PZ / 1656 / AR EP 1 907 553 B1

Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant

Physiol 87: 671-674 (soybean); Finer & McMullen (1991) In Vitro Cell Dev Biol 27P: 175-182 (soybean); Singh et al. (1998) Theor Appl Genet 96: 319-324 (soybean); Datta et al. (1990)

Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc Natl Acad Sci USA 85: 43054309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (corn); patents

United States of America 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);

Hooykaas-Van Slogteren et al. (1984) Nature 311: 763-764; U.S. Patent

Well. 5,736,369 (cereals); Bytebier et al. (1987) Proc Natl Acad Sci USA 84: 5345-5349 (lily); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)

Plant Cell Rep 9: 415-418) and Kaeppler et al. (1992) Theor Appl Genet 84: 560-566 (mustache transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Rep 12: 250-255; Christou & Ford (1995)

Annals of Botany 75: 407-413 (rice) and Osjoda et al. (1996) Nat Biotechnol 14: 745-750 (maize with Agrobacterium tumefaciens).

[0029] Alternatively, polynucleotides can be introduced into plants by contacting the plants with the virus or viral nucleic acids. Generally, these methods include embedding the polynucleotide into a viral DNA or RNA molecule. It is believed that the desired polypeptide may be initially synthesized as part of the viral polyprotein, which may later be processed by in vivo or in vitro proteolysis, resulting in the production of the desired recombinant protein.

In addition, promoters are also considered to include those used for transcription by viral RNA polymerases. Techniques for introducing polynucleotides into plants and expressing the protein they encode in which viral DNA or RNA molecules participate are known, see, for example, patents

United States of America 5,889.191, 5,889.190, 5,866,785, 5,589,367 and 5,316,931.

[0030] Transient transformation methods include, but are not limited to, the introduction of polypeptides, such as a recombinase protein, directly into the body, the introduction of polynucleotides, such as DNA and / or RNA polynucleotides, and the introduction into the body of an RNA transcript, such as mRNA encoding recombinase. Such methods include, for example, microinjection or molecular bombardment. See, for example, Crossway et al. (1986) Mol Gen Genet 202: 179-185;

Nomura et al. (1986) Plant Sci 44: 53-58; Hepler et al. (1994) Proc Natl Acad Sci USA

91: 2178-2180 and Hush et al. (1994) J Cell Sci 107: 775-784.

[0031] Cells having the introduced sequence can be grown to plants in accordance with conventional methods, see, for example, McCormick et al. (1986)

Plant Cell Rep 5: 81-84. These plants can be grown and pollinated with the same transformed variety or other variety, and then the obtained progeny expressing the desired phenotypic features and / or containing the introduced polynucleotide or polypeptide are identified. Two or more generations can be grown to ensure stable maintenance and inheritance of the polynucleotide, after which their seeds are harvested. In this way, transformed seeds are also provided, also called transgenic seeds, containing a polynucleotide, for example, containing a modified FRT site, stably integrated into their genome.

[0032] Examples of suitable plant genera and species include, but are not limited to, monocotyledons and dicotyledons, such as maize (Zea mays), Brassica sp. (E.g., B. napus, B. rapa, B. juncea), especially those Brassica species useful as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), common millet (Panicum miliaceum), chumiza (Setaria italica), korakan (Eleusine coracana)), sunflower (Helianthus annuus), saflor (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum) potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple ( Pineapple 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, locust beans, fenugreek, garden beans, Chinese beans, gold beans, crescent beans, bean seeds, edible lentils, chickpeas and chestnut, Arabidopsis, vegetables, decorative plants, grasses, conifers; crops and cereals that provide the right seeds, oilseeds and other legumes. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), crescent beans (Phaseolus limensis), peas (Lathyrus spp.) And members of the genus Cucumis, such as cucumber (C. sativus) , cantaloupe (C.

cantalupensis) and musk melon (C. melo). Decorative plants include azaleas

PZ / 1656 / AR EP 1 907 553 B1 (Rhododendron spp.), Hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), cloves (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemums. Conifers include, for example, pines such as taeda pine (Pinus taeda), twisted pine (Pinus elliotii), yellow pine (Pinus ponderosa), dune pine (Pinus contorta) and California pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); western Christmas tree (Tsuga canadensis); Sitka Spruce (Picea glauca); redwood (Sequoia sempervirens); real firs such as silver fir (Abies amabilis) and balsam fir (Abies balsemea); and cedars, such as giant thuja (Thuja plicata) and nutka cypress (Chamaecyparis nootkatensis).

[0033] The term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, regenerative creative tissue, plant clumps and plant cells that are entirely in plants or parts of plants, such as embryos, pollen , ovules, seeds, flowers, kernels, ears, cobs, husks, stems, roots, root tips, anthers and the like.

[0034] Prokaryotic cells may also be used in the method. Prokaryotic cells include various E. coli strains; however, strains of other bacterial microorganisms 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. These 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 (ATCC53323), JM110 (ATCC 47013), LE392 (ATCC 33572), K802 (ATCC 33526) , NM522 (ATCC 47000), RR1 (ATCC31343), 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: 557-580, Schatz et al. (1989) Cell 59: 1035, Bullock et al. (1987) BioTechniques 5: 376-378, ATCC Bacteria and Bacteriophages (1996) 9th edition and Palmer et al. (1994)

Gene 143: 7-8.

[0035] For example, but not limited to, viral strains include, but are not limited to, the genus geminivirus, begomovirus, curtovirus, mastrevirus, viruses

(-) strand RNA, (+) strand RNA viruses, potyvirus, potexvirus, tobamovirus or other DNA viruses, nanoviruses, viroids 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), barley mosaic virus (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 striped geminivirus (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 mosaic virus (TGMV) (Elmer et al. (1990) Nucleic Acids Res 18: 2001-2006) and wheat stunting virus (WDV) (Woolston et al. (1989) Nucleic Acids Res 17: 60296041) and their derivatives. See also, Porat et al. (1996) Mol Biotechnol 5: 209-221.

[0036] Commonly used prokaryotic control sequences include promoters for transcription initiation, optionally with an operator, together with ribosome binding sequences, include such commonly used promoters as the betalactamase (penicillinase) and lactose (iac) promoter system (Chang et al. (1977) Nature

198: 1056), tryptophan promoter system (trp) (Goeddel et al. (1980) Nucleic Acids

Res 8: 4057) and the lambda phage LP promoter and ribosome N gene binding site (Shimatake et al. (1981) Nature 292: 128).

[0037] The vector is selected to allow insertion into the appropriate host cell. Bacterial vectors are usually of plasmid or phage origin. Suitable bacterial cells are infected with phage vector molecules or bacterially transfected using "naked" phage DNA vector.

If a plasmid vector is used, bacterial cells are transfected with the plasmid DNA vector. Prokaryotic / bacterial expression systems are available for protein expression 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.

[0038] For expression of the desired polynucleotide, the use of various eukaryotic expression systems, such as yeast, insect cell lines, plant cells and mammalian cells is known. In some examples, transformed / transfected plant cells are used as expression systems. The synthesis (introduction / expression) of a heterologous nucleotide sequence in yeast is well known (Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor

Laboratory). Saccharomyces cerevisiae and Pichia pastoris are two species of yeast widely used for the production of eukaryotic proteins. Vectors, strains and

Expression protocols in Saccharomyces and Pichia are known and commercially available (e.g.,

InVitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and, if preferred, an origin of replication, termination sequences and the like.

[0039] Recombinant baculoviruses are obtained by inserting specific, interesting sequences into the baculovirus genome using known protocols, using commercially available vectors and reagents (e.g.,

InVitrogen, Life Technologies Incorporated). Commercially available vectors are readily obtained with various promoters such as polyhedrins and p10, optional signal sequences for protein secretion or affinity sequences such as the 6X histidine tag sequence. Such recombinant viruses are cultured, maintained and propagated in commercially available cell lines from several insect species, including Spodoptera frugiperda and

Trichoplusla nl. Insect cells can be cultured in a variety of media using well-defined protocols, for example, with or without the addition of bovine serum. The cultured cells are infected with recombinant viruses and the appropriate sequence is expressed. Proteins obtained as a result of expression in the baculovirus system have been characterized in detail and in many cases their post-translational modifications such as phosphorylation, acylation, etc. are identical to native protein.

[0040] A modified FRT recombination site of the invention in combination with

FRT1 can be used as a reagent in kits. Such kits may further comprise FLP recombinase and may also include a polynucleotide, optionally integrated into the genome of the body, comprising at least one target flanked by functional, different, non-recombined, modified FRT recombination sites. Instructions for use may be included with any kit.

[0041] Modified FRT recombination sites can be used in various in vitro and in vivo site-specific recombination methods that allow for targeted incorporation, exchange, modification, alteration, excision, reversal and / or expression of the desired nucleotide sequence, see for example .

WO99 / 25821, WO99 / 25854, WO99 / 25840, WO99 / 25855 and WO99 / 25853.

[0042] The methods use a site-specific recombination system.

A site-specific recombinase, also called a recombinase, is a polypeptide that catalyzes conserved site-specific recombination between its

In this respect, recombinase includes native polypeptides as well as variants and / or fragments that retain activity, and native polynucleotides and variants and / or fragments that encode recombinase that retains activity. The recombinase used in the methods can be native recombinase or a biologically active fragment or variant of recombinase.

The native polypeptide or polynucleotide contains a naturally occurring amino acid sequence or nucleotide sequence. For a review 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 integrase and resolvase recombinases, their biologically active variants and fragments, and any other, naturally occurring or recombined enzymes or variants that catalyze conserved, site-specific recombination between specific recombination DNA sites .

[0043] The integrase recombinase group includes over one hundred members and includes, for example, FLP, Cre, lambda integrase 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, bacteriophage phi C31 from Streptomyces (Kuhstoss et al. (1991) J

Mol Biol 20: 897-908); site-specific recombination system SSV1 with Sulfolobus shibatae (Maskhelishvili et al. (1993) Mol Gen Genet 237: 334-342) and an integration system based on retroviral integration (Tanaka et al. (1998) Gene 17: 67-76).

Some recombinases do not require cofactors or a supercoiled substrate. Such recombinases include native Cre (SEQ ID NO: 45 and 46), native FLP (SEQ ID

NO: 48 and 49) or active variants or fragments thereof (SEQ ID NO: 47 and 50).

[0044] FLP recombinase was used in the method of the invention. recombinase

FLP is a protein that catalyzes a site-specific reaction that is involved in duplicating the copy quantity of a two-micron plasmid from S. cerevisiae during replication

GOUT. 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). FLP recombinase for use in methods and with compositions may be of the genus Saccharomyces. It is also possible to synthesize a recombinase containing polynucleotide using plant-friendly codons for optimal expression in the appropriate plant. A recombinant FLP enzyme encoded by a nucleotide sequence containing preferred maize-derived codons (FLPm) is known that

PZ / 1656 / AR EP 1 907 553 B1 catalyzes the occurrence of site-specific recombination (SEQ ID NO: 50 and patent

United States of America 5,929,301). Additional functional variants and fragments are known

FLP (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).

[0045] 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: 1509-1514; Chen et al.

(1996) Somat Cell Mol Genet 22: 477-488; Shaikh et al. (1977) J Biol Chem 272: 56955702 and Buchholz et al. (1998) Nat Biotechnal 16: 617-618. Cre nucleotide sequences can also be synthesized using plant-friendly codons, for example such sequences (moCre) are described in WO 99/25840 and listed in SEQ ID NO: 47.

[0046] Chimeric recombinase is a fusion recombinant protein capable of catalyzing site-specific recombination between recombination sites that are derived from different recombination systems. Methods for making and using such chimeric recombinases or active variants or fragments thereof are described in WO 99/25840. Analysis of recombinase activity is known and generally involves measuring the total enzyme activity on DNA substrates containing recombination sites. For example, to measure 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.

Such analysis is described in Vetter et al. (1983) PNAS 80: 7284. Alternatively, DNA excision from the molecule or intermolecular recombination frequency induced by the enzyme can be analyzed, 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 analyzed by cleavage of the flanked sequence by recombinant FRT sites, resulting in measurable activation of the marker gene. Similar strategies can be used for Cre or other recombinase enzymes.

[0047] Polynucleotide variants and proteins also include sequences and proteins resulting from mutagenesis and / or recombination procedures, such as shift

GOUT. By such a procedure, one or more different recombinase coding sequences can be used to create a new recombinase protein with the desired properties. In this way, recombinant libraries are generated

Polynucleotides from a population of related polynucleotides containing sequence regions that exhibit significant sequence identity and may undergo homologous recombination in vitro or in vivo. PZ / 1656 / AR EP 1 907 553 B1 Strategies for such DNA shift are known and include, for example, Stemmer (1994) Proc Natl Acad Sci USA 91: 1074710751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nat Biotech 15: 436438; 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 patents

United States of America 5,605,793 and 5,837,458.

[0048] The methods and compositions utilized the modified FRT sites disclosed in the invention, FRT12 (SEQ ID NO: 21), in combination with FRT1 (SEQ ID NO:

39). The recombination site is any native or synthetic / artificial polynucleotide that is recognized by the appropriate recombinase enzyme.

Many recombination systems are known in which the appropriate recombination site (s) are used with the appropriate system, 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 usable recombination sites are known, including FRT sites including the native FRT site (FRT1, SEQ ID NO: 39) and various functional FRT variants, 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 other functionally modified sites

FRT. See, for example, WO 03/054189, WO 02/00900, WO 01/23545 and Schlake et al. (1994) Biochemistry 33: 12745-12751.

[0049] The 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 site activity of the LOX variant is presented in Lee et al. (1998) Gene 216: 55-65 and in US Patent

6,465,254. Also, see, for example, Schlake & Bode (1994) Biochemistry 33: 1274612751; Huang et al. (1991) Nucleic Acids Res 19: 443-448; Sadowski (1995) W.

Progress in Nucleic Acid Research and Molecular Biology Vol. 51, pp. 53-91; patent

United States of America 6,465,254; Cox (1989) In Mobile DNA, Berg and Howe (ed.) American

Society of Microbiology, Washington DC, pp. 116-670; Dixon et al. (1995) Mol

Microbiol 18: 449-458; Umlauf & Cox (1988) EMBO J 7: 1845-1852; Buchholz et al.

(1996) Nucleic Acids Res 24: 3118-3119; Kilby et al. (1993) Trends Genet 9: 413-421;

Rossant & Geagy (1995) Nat Med 1: 592-594; Albert et al. (1995) Plant J 7: 649-659;

Bayley et al. (1992) Plant Mol Biol 18: 353-361; Odell et al. (1990) Mol Gen Genet

PZ / 1656 / AR EP 1 907 553 B1

223: 369-378; Dale & 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.

[0050] Any suitable recombination site or set of recombination sites may be used in methods and compositions in addition to the FRT12 / FRT1 combination of the invention, including the FRT site, functional FRT site variant, LOX site and functional LOX site variant, any combination thereof or any another combination of known recombination sites.

[0051] The term "directly repeated" indicates that the recombination sites in the set of recombinogenic recombination sites are arranged in the same orientation, such that recombination between these sites results in cleavage rather than inversion of the participating DNA sequence. The term "inverted" recombination site (s) indicates that the recombination sites in the set of recombinogenic recombination sites are in the opposite orientation, such that the recombination between these sites results in a reversal rather than a cleavage of the participating DNA sequence.

[0052] The target site and transfer cassette used in the method of the invention include a first recombination site containing SEQ ID NO: 21 and a second recombination site containing SEQ ID NO: 39. The local-specific recombinase (s) used will be FLP recombinase or an active variant thereof will be provided. In the same way, when Lox sites are also used, Cre recombinase or an active fragment thereof can also be used. If the set of functional recombination sites contains both FRT12 and FRT1 sites and a Lox site, FLP / Cre chimeric recombinase or its active variant or both FLP and Cre recombinases or their active variants can be used.

[0053] The disclosure includes any method that allows the polypeptide and / or polynucleotide such as recombinase, target site, transfer cassette, desired polynucleotide to be placed together with said components. For example, it is possible to provide a cell with various components by various methods, including a transient and stable transformation method; simultaneous introduction of DNA, mRNA or protein recombinase directly into the cell; using an organism, cell, strain or line that expresses recombinase to initiate transformation; or the growth / culture of the target transferring organism and crossing it with an organism that expresses active recombinase protein and selection of transformed offspring. Any promoter may be used to express the appropriate recombinase, including constitutive, induced, developing

A temporal or spatial structure-driven promoter capable of regulating expression in a suitable organism.

[0054] Compositions comprising a recombinogenic, modified FRT recombination site are provided. The polynucleotide of the invention comprising a recombinogenic modified FRT recombination site, FRT12 (SEQ ID NO: 21) and wild type FRT1 (SEQ ID NO: 39) can be used in local-specific recombination methods.

[0055] Methods may use targets and transfer cassettes to manipulate, exchange, cut, alter, invert and / or introduce the nucleotide sequence in vivo or in vitro. The destination site contains at least one recombination site. In specific examples, the target comprises a polynucleotide that is directly flanked by at least two recombination sites, including sets of functional recombination sites that are different and non-recombinogenic with respect to each other;

corresponding and recombinogenic with respect to each other or different and recombinogenic with respect to each other. One or more of the sequences involved may be between recombination sites of the target.

Particularly interesting sequences involved in the reaction include linker sequences, adoptor sequences, regulatory regions, introns, restriction sites, enhancer sequences, separation sequences, selection markers, desired nucleotide sequences, promoters and / or other sites that are useful in construction or analysis vector. In addition, it is contemplated that a recombination site may be contained in the desired nucleotide sequence, including introns, coding sequences, 5 'UTR, 3' UTR and / or regulatory regions.

[0056] In specific examples, the target is in 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 believed that a cell or organism may contain multiple targets that can be located at one or more loci on or within chromosomes. Multiple, independent manipulations of each destination in the body are possible. In addition, the target site may also contain an expression cassette comprising a nucleotide sequence encoding the appropriate recombinase. In another example, the nucleotide sequence encoding the recombinase is stably integrated into the genome of the body.

[0057] The methods also use transfer cassettes. The transfer cartridge contains at least one recombination site. In specific examples, the transfer cassette containing the polynucleotide is flanked by at least the first recombination site and the second recombination site, wherein 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 the second site. In the event that the target site and the transfer cassette containing compatible recombination sites and the recombinase are combined, the nucleotide sequence between the recombination sites of the destination site will be exchanged with the nucleotide sequence between the recombination sites of the transfer cassette. The term "flanked by" when used in reference to the location of recombination sites of a destination or transfer cassette, refers to the position immediately adjacent to the sequence that is considered to be replaced or inserted.

[0058] The transfer cassette may further comprise a desired polynucleotide. The recombination sites may be directly adjacent to the desired polynucleotide or one or more participating sequences may be contained between one or both ends of the desired polynucleotide and the recombination sites. Particularly interesting sequences involved in the reaction include linker sequences, adapter sequences, enhancer sequences, introns, spacer sequences, restriction sites, selectable markers, desired polynucleotides, promoters and / or other sites that are useful in constructing or analyzing the vector. Recombination sites may be included in the polynucleotide of interest, including introns, coding sequences and / or 5 'and 3' untranslated regions.

[0059] In the method of the invention, the transfer cassette and target contain a functionally modified FRT recombination site containing SEQ ID

NO: 21 and a second recombination site containing SEQ ID NO: 39.

[0060] Any means can be used to bring together the various components of the recombinant system. For example, in in vitro systems, recombinase and polynucleotide (s) containing recombination sites can be provided by contacting the components under appropriate conditions to allow recombination. Alternatively, various methods are known for introducing nucleotide sequences and polypeptides into an organism, including, for example, transforming and introducing a polypeptide, DNA or mRNA into a cell.

PZ / 1656 / AR EP 1 907 553 B1

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 sequence from the mRNA mosaic virus protein coat of the mosaic virus alpha alpha (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625);

tobacco mosaic virus (TMV) leader sequence (Gallie et al. (1989) in Molecular

Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and the leader sequence of the corn variegated virus (MCMV) (Lommel et al. (1991) Virology 81: 382-385).

See also Della-Cioppa et al. (1987) Plant Physiol 84: 965-968. Other methods or sequences known to enhance translation may also be used, for example, introns and the like.

[0095] When preparing the DNA construct, the various DNA fragments can be manipulated to place the sequence in the correct orientation and, if necessary, in the correct reading frame. To this end, adapter or linker sequences may be used to link DNA fragments, or other manipulations may be performed to obtain preferred restriction sites, removal of unnecessary DNA, removal of restriction sites, or the like. To this end, in vitro mutagenesis, primer repair, cleavage, annealing, re-substitution, transfer and / or transversions may be used.

[0096] Generally, the DNA construct will contain a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues and are discussed in detail herein, as are exemplary suitable promoters.

[0097] The following examples are provided for illustrative purpose and in no way limit the present invention.

EXPERIMENTAL PART

Reference example 1. Preparation of libraries containing modified FRT recombination sites [0098] Two complementary degenerate oligonucleotides containing FRT sequences with 6 central linker positions, randomly mutated: oligo 1: 5'gccagcatgcaagctcgccNcccNcccNcccNcccNcccNcccNcccNccNccNccNccNccNccNccNccNccNcccNccNccNccNcccNccNccNccNccNTcNTcT -3 '(SEQ ID NO: 52) and

Oligo2: 5'-cgttccgcggatccagatctcgaagttcctattctNNNNNagta taggaacttcggaattcaagcttgcatgctggc-3 '(SEQ ID NO: 53).

[0099] The connecting area is 8 bp. In this experiment, the central region of 6 bp was modified, and therefore, another two nucleotides remained unchanged. One pmol of oligonucleotide 1 and one pmol of oligonucleotide 2 were hybridized (annealing) using heat denaturation at 95 ° C for 2 minutes, followed by gradual cooling to room temperature.

The attached oligonucleotides were digested with EcoRI and BamHI and ligated at EcoRI / BamHI sites of the pSportl derivative vector, so that 3 additional bases formed an HpaI restriction site (BRL Life Technologies, Gaithersburg, MD), and vector PHP13273 containing the spectinomycin resistance gene allowed for two plasmid modified FRT libraries. A molar ratio of attached oligonucleotides to pSport of 10: 1 and 4: 1 was used, a

PHP13273 was used in the appropriate ligation reactions. Under such ligation conditions, 10 of 10 randomly collected colonies contained a monomer insertion of the modified FRT site.

The modified FRT library called "library A" is located in the pSport vector and carries an antibiotic ampicillin resistance marker. The modified FRT library called "library B" is located in the PHP13273 vector and carries the antibiotic marker spectinomycin resistance. A total of 15,904 colonies were collected to create the FRT A library, and to create a B library

FRT collected 19,600 colonies. The cover covers 4 central positions 4 (46= 4096) in place of FRT. Plasmid DNA was isolated from these two libraries and used for screening library scaling.

Reference example 2. Screening library scaling to identify recombined modified FRT recombination sites [0100] Equimolar amount of DNA from each modified FRT library A and B was mixed in one tube containing in vitro recombination reaction buffer with FLP. Typically, 20μ1 recombination reaction buffer containing 25mM Tris / Cl pH 7.5, 10mM MgCl2, 5mM DTT, 50fmol of library A DNA, 50fmol of library B

DNA and 2μ1 FLP (final concentration 0.07μg / μl). The reaction was carried out at 30 ° C and samples were taken at various time points. At each time point, 2 μ1 was taken and the reaction stopped by boiling for 1 min with gradual cooling to room temperature. Typically, samples were taken at 0,

2, 5, 10, 30, 60 and 90 minutes and they could be analyzed for rapid determination

PZ / 1656 / AR EP 1 907 553 B1 vs slow response at FRT sites. When only one time point was taken into account, the time point after 90 minutes was used.

[0101] Reaction samples were transferred to E. coli DH5a cells according to standard procedures. Equal amounts of each transformation reaction mixture were spread on one plate, each containing only ampicillin, only spectinomycin, or containing both ampicillin and spectinomycin. DNA with double sequence insertion, obtained as a result of successful recombination, will contain both selectable markers and, therefore, after transfer to E. coli, it will provide resistance to both ampicillin and spectinomycin.

[0102] Such colonies exhibiting resistance to both antibiotics were harvested and plasmid DNA was obtained using a 96-well HTP plasmid DNA preparation kit (Millipore, Billerica, MA USA). Potential FRT sites were obtained by PCR using primers flanking recombinant FRT sites in DNA with double sequence insertion. The PCR primers used were as follows, a 5 'end hybridizing primer, so-called forward / forward sense strand: 5'gcacatacaaatggacgaacgga-3 (SEQ ID NO: 54) and 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 s; 61 ° C, 2 min; one cycle: 67 ° C, 3 min; leaving at: 4 ° C. The sequence of the amplified potential FRT sites was determined by sequencing using cyclic sequencing (essentially as described in Slatko et al. (1993) DNA Sequencing. In Current Protocols in Molecular Biology, ed.

By Ausubel et al.) Roz. 7, pp. 7.0.1-7.6.13. New York: John Wiley & Sons).

Example 3. Methods for analyzing the cleavage efficiency of recombined modified FRT recombination sites [0103] To examine the cleavage efficiency of a recombinant potential FRT site, excision vectors were prepared into which two copies of the potential recombined FRT site recombined in the orientation directly flanking the sequence were cloned maize ubiquitin promoter in pSport (BRL Life Technologies, Gaithersburg, MD). The excision reaction was carried out under the following conditions: 3μ1 miniprep DNA excision vector (2mg / ml), 1μ1 10x buffer (250mM Tris Cl pH 7.5, 100mM MgCl2, 50mM DTT), 5μΙ ddH2O and 1μΙ 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 using EcoRV and XhoI, and then analyzed by agarose gel electrophoresis.

[0104] EcoRV produces a single cut in the pSport vector backbone, while XhoI produces a single cut in the maize ubiquitin promoter sequence. Double digestion of the non-recombinant excision vector results in two 4332 bp and 769 bp fragments. Double digestion of the product vector after excision results in an additional 952 bp fragment.

DNA fragments were counted using the Quantity One program from Bio-Rad Laboratories. When the excision occurs, an increased amount of the 952 bp fragment is obtained and less fragment 769 is obtained. Therefore, the ratio of the 952 bp fragment to 769 bp fragment allows the absolute performance of the notch to be measured. In this experiment, the relative recombination excision efficiency (% excision efficiency) of the FRT site was calculated as the excision efficiency in the presence of native yeast

FLT of the first modified FRT site with the second modified site

FRT divided by the cleavage efficiency of the FRT wild pair (SEQ ID

NO: 39) X 100%.

[0105] Various modified FRT recombination sites identified by the method of Example 2 were analyzed for their ability to retain biological activity. Table 1 shows the various functional, modified FRT recombination sites and their relative recombination efficiency determined as described above. Data for FRT sites other than FRT1 and FRT12 are for reference purposes only.

Table 1

<td>Places FRT</td><td>SEQ ID NO of the minimal modified space FRT</td><td>Sequence liaison</td><td>SEQ ID No. modified linker sequence</td><td>Cut out 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>

PZ / 1656 / AR EP 1 907 553 B1

<td>Places FRT</td><td>SEQ ID NO of the minimal modified space FRT</td><td>Sequence liaison</td><td>SEQ ID No. modified linker sequence</td><td>Cut out efficiency (%)</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">* Linker sequences were flanked by the wild-type 13 base pair symmetrical element mentioned in Figure 1.</td>

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Example 4. Methods for analyzing the efficiency of jointly inserting sequences for modified recombination recombinant FRT sites [0106] The experiment was performed as described in Example 2. In brief, FRT1, 5 and 6 (SEQ ID NO: 39, 40 and 41) were cloned individually in EcoRI / BamHI sites in PHP13273 and the vector pSport1 was modified. 50 fmol DNA from FRT1 in PHP13273 (Spec ') was mixed with 50 fmol DNA from FRT1 in modified pSport1 (Ap') in 20μ1 reaction buffer containing 25mM Tris Cl at pH 7.5, 10mM MgCl2, 5mM DTT and 2μ1 FLP (final concentration 0.07μg / μl). At each time point, a 2 μ1 sample was taken and the reaction stopped by gradually cooling to room temperature for 1 min. Samples with the reaction mixture were transferred to E. coli DH5a cells according to standard procedures.

Equal amounts of each transformation reaction mixture were spread on one plate, each containing only ampicillin, only spectinomycin, or containing both ampicillin and spectinomycin. Double-insertion DNA obtained through successful recombination via the FRT1 site will carry both selection markers, and therefore, when transferred to E. coli, will provide resistance to both ampicillin and spectinomycin. Colonies exhibiting resistance to both antibiotics were collected and further analyzed for double insertion plasmid DNA. Clones that were resistant to both antibiotics but did not contain double-inserted plasmid DNA were not included in the calculation of the double-insertion frequency.

[0107] The frequency of FRT1 double insertion was determined by calculating the percentage of colonies containing double-inserted plasmid DNA from colonies resistant to one antibiotic. Similarly, FRT5 or FRT6 insertion was performed in vitro and the frequency of FRT5 or FRT6 double insertion was determined, respectively. The results are shown in Table 2. Data for FRT5 and FRT6 are included for comparison purposes only.

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Table 2. Percentage of double insertion obtained by in vitro recombination with participation (%)

[0108] In vitro recombination with FLP was performed as previously described. When DNA containing different FRT sites is mixed in the reaction, as in the previously described library scaling screen, intermolecular recombination occurring between two corresponding FRT sites is further reduced. In reactions containing only FRT1 sites, only FRT5 sites or only FRT6 sites, 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).

[0109] In this example, plasmid DNA containing three different FRT sites (FRT1, FRT5 and FRT6) were mixed in the reaction, each located in modified pSport1 carrying the A selection markerr and PHP13273 carrying Specr. Among FRT1, FRT5 and FRT6, two different FRT sites do not recombine with each other. In a reaction containing an equimolar amount of DNA containing FRT, FRT1, FRT5 and FRT6 sites, recombination efficiency between any two corresponding FRT sites is reduced. The results are shown in Table 3. Data corresponding to FRT5 and FRT6 are for comparative purposes only. The combined frequency of joint insertion occurring between two FRT1 sites, two FRT5 sites and two FRT6 sites was 0.09% after 90 minutes, approximately 10 times less than for a reaction only taking place for FRT1 site. Most products containing a common insertion are derived from recombination using the most efficient FRT sites, FRT1, in reaction, as evidenced by the fact that all of the 10 randomly collected products containing the common insertion were products of recombination of FRT1 sites. In the reaction involving the lower molar amount of DNA containing the FRT1 site (molar ratio between FRT1, FRT5 and FRT6 is: 0.04: 1.00: 1.00), the total level of recombination has continued

PZ / 1656 / AR EP 1 907 553 B1 lowering. In addition, none of the 10 randomly collected products containing the co-insertion were FRT1 site recombination products.

Table 3

<td>FRT seats *</td><td>Product containing a joint insertion (%)</td><td>Analysis of the product containing the joint insertion FRT1 / total product containing the joint insertion</td>

<td>FRT1 (Apr. 50fmol) + FRT1 (Specr, 50fmol)</td><td> 0.98</td><td> 10/10</td>

<td>FRT5 (Apr. 50fmol) + FRT5 (Specr, 50fmol)</td><td> 0.08</td><td>ON</td>

<td>FRT6 (Apr. 50fmol) + FRT6 (Specr, 50fmol)</td><td> 0.28</td><td>ON</td>

<td>FRT1 (Apr. 16fmol) + FRT1 (Specr, 16fmol) + FRT5 (Apr. 16fmol) + FRT5 (Specr, 16fmol) + FRT6 (Apr. 16fmol) + FRT6 (Specr, 16fmol)</td><td> 0.09</td><td> 10/10</td>

<td>FRT1 (Apr. 0.8fmol) + FRT1 (Specr, 0.8fmol) + FRT5 (Apr. 20fmol) + FRT5 (Specr, 20fmol) + FRT6 (Apr. 20fmol) + FRT6 (Specr, 20fmol)</td><td> 0.07</td><td> 0/10</td>

<td colspan="3">* The selection marker and molar amount of DNA used in the reaction are given in brackets.</td>

Example 5: Transformation of plants

A. Transformation by particle bombardment and regeneration of maize callus tissue [0110] Immature maize embryos from the greenhouse or donor plants from the High II crop field (hill) were bombarded with an isolated polynucleotide

Containing a recombination site, transfer cassette, target site and / or recombinase, provided in the context of the present invention. If the polynucleotide does not include a selectable marker, another polynucleotide containing the selectable marker gene may be simultaneously precipitated on the molecules used for the bombardment. For example, a plasmid containing the PAT gene may be used (Wohlleben et al. (1988) Gene 70: 25-37), which provides resistance to bialafos herbicide. The transformation was performed as below.

[0111] The ears were surface sterilized in 50% Chlorox chlorine bleach plus 0.5% Micro detergent for 20 minutes and washed twice with sterile water. The immature embryos were excised and placed on the embryo axis at the bottom (upside down), 25 embryos per plate. They grew in the dark, in 560L agar medium for 4 days before bombardment. 560L medium is an N6-based medium containing Eriksson vitamins, thiamine, sucrose, 2,4-D and silver nitrate. On the day of the bombing, the embryos were transferred for 4 hours to 560Y medium and placed in 2.5-cm target zones. 560Y medium is a highly osmotic medium (560L with high sucrose concentration).

[0112] A plasmid vector containing the desired polynucleotide operably linked to the selected promoter was constructed. This plasmid DNA, plus, if necessary, plasmid DNA containing the PAT selection marker, was precipitated on 1.0 μm (average diameter) of gold pellets using the following CaCl precipitation procedure2: 100 μΐ of prepared golden particles (0.6 mg) in water, 20 μΐ (2 μg) DNA in TrisEDTA buffer (1 μg in total), 100 μΐ 2.5 M CaCl2, 40 μΐ 0.1 M spermidine.

[0113] Each reagent was added sequentially to the gold particle suspension. The final mixture was briefly sonicated. After the precipitation time, the tubes were briefly centrifuged, the liquid removed, washed with 500 μΐ 100% ethanol and again centrifuged for 30 seconds. The liquid was removed again and 60 pL 100% ethanol was added to the final pellet of gold particles. In particle bombardment using gene arrow, the gold / DNA molecules were briefly sonicated and 5P 5 spot applied to the center of each macrocarrier and allowed to dry for about 2 minutes before bombardment.

[0114] Sample plates were bombarded from a distance of 8 cm from the retaining screen to the tissue using a biohistic handle with he ^ DuPont particles. All samples received a single shot of 650 PSI, in total, in ten portions taken from each tube with prepared particles / DNA.

[0115] Four to 12 hours after bombardment, the embryos were transferred to 560P (low osmotic medium initiating the growth of caustic tissue, similar to a 560L lower ate

(Nitrate content), for 3-7 days, then transferred to a 560R selection medium, N6-based medium similar to 560P containing 3 mg / liter bialafos and grown for 2 weeks. After approximately 10 weeks of selection, callus tissue clone samples were taken for PCR and / or analysis of the desired polynucleotide activity. Positive lines were transferred to 288J medium, an MS-based medium with a lower sucrose content and a lower hormone level to initiate plant regeneration. After somatic maturation of the embryos (2-4 weeks), well-developed somatic embryos were transferred to the germination medium and placed in an illuminated growing room. Approximately 7-10 days later, the growing seedlings were transferred to the ground in test tubes for 7-10 days until the seedlings developed well. The plants were then transferred to flat vessels (equivalent to 2.5 "pots) containing potting soil and grown for a week in the growing room, then grown for an additional 1-2 weeks in a greenhouse, after which they were transferred to Classic ™ 600 pots (1.6 gallons) and grown to maturity. Plants were monitored for expression of the desired polynucleotide.

B. Agrobacterium-mediated transformation and regeneration of maize callus tissue [0116] For the transformation of maize, using Agrobacterium, the Zhao method (US Patent 5,981,840) provided in the present invention polynucleotide containing a recombination site, transfer cassette, site target and / or recombinase.

[0117] Briefly, immature embryos were isolated from maize and contacted with an Agrobacterium suspension containing the desired polynucleotide, wherein the bacteria were able to transfer the desired nucleotide sequence to at least one cell of at least one immature embryo (stage 1: infection stage). At this stage, the immature embryos were immersed in an Agrobacterium suspension to initiate inoculation. The embryos grew together with Agrobacterium for some time (stage 2: stage of co-culture). After this breeding time, an optional "rest" stage (stage 3: rest stage) can be carried out. Immature embryos were grown on a solid medium with an antibiotic, but without the selection agent used to eliminate Agrobacterium, and in the rest phase for infected cells. Then, inoculated embryos were cultured in medium containing the selection factor and harvested transformed callus tissue was harvested (stage 4: selection stage). Immature embryos were grown on a solid medium with a selection factor,

PZ / 1656 / AR EP 1 907 553 B1 resulted in the selective growth of transformed cells. Plants were then obtained from callus tissue (stage 5: regeneration stage) and those that grew on the selective medium were grown on solid medium to obtain plant regeneration.

C. Transformation of dicotyledonous plants [0118] The polynucleotide provided in the present invention comprising a recombination site, transfer cassette, target site and / or recombinase can be introduced into a soybean embryo suspension, by bombardment using essentially the methods described in Parrott et al. (1989) Plant Cell Rep. 7: 615-617. This method, along with the modifications, are described below.

[0119] Seeds were removed from the pots when the cotyledons were between 3 and 5 mm long. The seeds were sterilized in a bleach solution (0.5%) for 15 minutes, after which the seeds were washed with sterile distilled water. Immature cotyledons were excised by removing part of the seed that contained the embryo axis. The cotyledons were then removed from the seed shells by gently pushing the distal end of the seed with the blunt end of the scalpel blade. Then, cotyledons were placed on Petri dishes (flat side up) with SB1 initiation medium (MS salts, vitamins B5, 20 mg / L 2,4-D,

31.5 g / L sucrose, 8 g / L TC agar, pH 5.8). Petri dishes were incubated in the light (16 h per day; 75-80 μΕ) at 26 ° C. After 4 weeks of incubation, cotyledons were transferred to fresh SB1 medium. After an additional two weeks, globular somatic embryos that showed areas of proliferation were excised and transferred to FN Lite fluid medium (Samoylov, et al. (1998) In Vitro Cell Dev. Biol.-Plant 34: 8-13). About 10 to 12 small clusters of somatic embryos were placed in 250 ml bottles containing 35 ml SB172 medium. The soybean embryogenic suspension culture was maintained in 35 mL liquid medium on a rotary shaker, at 150 rpm, at 26 ° C, under fluorescent light (20 μΕ) on a 16: 8 hour day / night schedule. Cultures were refreshed every two weeks by inoculating approximately 35 mg of tissue in 35 mL of liquid medium.

[0120] Cultures of embryogenic suspension from soybean were then transformed using a particle bombardment shotgun (Klein et al. (1987) Nature 327: 70; US Patent No. 4,945,050). The BioRad Biolistica PDS1000 / HE apparatus can be used for these transformations. The marker gene used to facilitate soybean transformation is the chimeric gene consisting of the 35S promoter from 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

PZ / 1656 / AR EP 1 907 553 B1

25: 179-188) and the 3 'region of the nopaline synthase gene from the T-DNA of the Ti plasmid from Agrobacterium tumefaciens.

[0121] To 50 μL of 60 mg / mL 1 μm suspension of golden particles (in order) was added: 5 μL DNA (1 μg / μL), 20 μΙ spermidine 0.1 M) and 50 μL CaCl2 (2.5 M). The particle preparation was shaken for three minutes, centrifuged in a microcentrifuge for 10 seconds and the supernatant removed. DNA-coated particles were washed once in 400 μL of 70% ethanol and resuspended in 40 μL of anhydrous ethanol. DNA suspension / molecules were sonicated three times one second. Five μL of DNA-coated gold particles were then applied to each macro carrier disk.

[0122] Approximately 300-400 mg of the bi-weekly culture suspension was placed on an empty, 60x15 mm Petri dish and pipette residual tissue was removed. The diaphragm rupture pressure was set to 1100 psi and the chamber was evacuated to a 28 inch mercury vacuum. The tissue was placed approximately 8 cm from the retaining screen and bombarded three times. After the bombardment, the tissue was halved and placed back into 35 ml FN Lite medium.

[0123] Five to seven days after the bombing, the liquid medium was replaced with fresh medium. Eleven days after the bombardment, the medium was replaced with fresh medium containing 50 mg / mL hygromycin. This selection medium was refreshed weekly. Seven to eight weeks after the bombing, green, transformed tissue growing from non-transformed, necrotic embryogenic clusters was noted. Isolated green tissue was removed and inoculated into individual bottles to generate new, clone-derived, transformed, embryogenic culture suspensions. Each new line was treated as an independent transformation event. Subsequent cultures were then prepared from suspensions and maintained as clusters of immature embryos, or as a result of maturation and germination of individual embryos, tissue was regenerated to the whole plant.

D. Isolation of DNA from callus and leaf tissue [0124] Presumed transformations can be screened for the presence of the transferred gene. Genomic DNA was extracted from callus tissue or leaves using a modified CTAB (cetyltriethylammonium bromide, Sigma H5882) method described by Stacey and Isaac (1994 In Methods in Molecular Biology Vol. 28, pp. 9-15, PG Isaac, Humana Press, Totowa, NJ). Approximately 100-200 mg of frozen tissue was pulverized in liquid nitrogen and homogenized in 1 ml CTAB extraction buffer (2% CTAB, 0.02 M EDTA, 0.1 M Tris-Cl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min in 65 ° C. Homogenized samples were allowed to cool in

PZ / 1656 / AR at room temperature for 15 min before performing a single protein extraction, using approximately 1 ml 24: 1 v / v chloroform: octanol. Samples were centrifuged for 7 min at 13,000 rpm and the upper layer of supernatant was collected using a wide tip pipette. DNA was precipitated from the supernatant by incubating in 95% ethanol in an ice bath for 1 h. DNA was wound on a glass hook, washed in 75% ethanol containing 0.2 M sodium acetate for 10 min, air dried for 5 min and resuspended in TE buffer. Five μΐ RNAse A were added to the samples and incubated at 37 ° C for 1 h. To count genomic DNA, gel electrophoresis was performed using 0.8% agarose gel in 1x TBE buffer. One microliter of each sample was fractionated along 200, 400, 600 and 800 ng μ1-1 λ of the undigested DNA marker.

Reference example 6. Comparison of the relative efficiency of recombination of different FRT sequences in maize cells [0125] Two analyzes are provided of measuring the relative level of activation of the transferred gene as a result of FLP excision, which causes the promoter and the transferred gene to be placed in a functional neighborhood. This method can be used to characterize the recombination efficiency of the corresponding and / or different recombination sites, and thus to determine whether the sites recombine or not recombine with other sites.

[0126] Two analyzes are discussed below: (A) assessing the activity of yellow fluorescent protein (YFP) in individual cells and (B) assessing luciferase activity.

A. Fluorescence analysis [0127] Three transgenic expression cassettes (listed in Table 4) were introduced in the tested FRT or control.

Table 4

<td>FRT construct under study</td><td>Control construct</td>

<td>Actin :: CFP :: nose</td><td>Actin :: CFP :: nose</td>

PZ / 1656 / AR EP 1 907 553 B1

YFP = yellow fluorescent protein; CFP = cyanorescent protein; CPN60 = maize chaparonine 60 promoter (Close (1993) Master's thesis, Iowa State University).

[0128] For both the control and assay with FRT, the relevant (or appropriate) three expression cassettes were mixed in equimolar ratio and introduced into thyroid immature Hi-II embryonic cells using standard molecule delivery methods. After two days, the number of cyano and yellow fluorescent cells was counted using a Leica epifluorescence stereomicroscope. Numerous cyanofluorescent cells were used to normalize the control and test sample, by providing relative measurement and determining how many cells the DNA received successfully to express transgenes. In order to check the correctness of this analysis system, FRT1 was used in the first experiment. As a control action, a mixture of the following three plasmids was used: Actin :: Cyan FP :: nos, CPN60: FRT1: YFP: 35s term and Ubi :: FLP :: pinII. In a control study in which the three plasmids mentioned above were co-introduced and two days later the number of cells with cyanofluorescence and yellow fluorescence were counted, it was expected that the number of these cells in the population would be approximately equivalent (1: 1).

[0129] In the test with FRT, when FRT1 was used in the notch activated excision cassette (CPN60: FRT1: GUS: FRT1: YFP: 35s term), it was expected that approximately 90-95% of the cells expressing cyanofluorescence also expressed yellow fluorescence, i.e. excision of the FRT1 flanking region is relatively efficient. Based on previous studies using FRT5, when FRT5 was used in the expression cassette, the incidence of cyanofluorescent cells that also expressed yellow fluorescent protein was expected to fall to approximately 15% of that observed with FRT1. [0130] The excision activated cassette can also be used to determine if two different FRT recombination sites can recombine or not recombine. To determine whether FRT1 and FRT5 are recombinogenic or non-recombinogenic relative to the second site, a notch activated cassette was constructed containing CPN60: FRT1: GUS: FRT5: YFP: 35s term. As indicated in Table 4, the three expression cassettes were mixed equimolarly and introduced into Hi-II thyroid cells of immature embryos using standard molecule delivery methods. After two days, cyano and yellow fluorescent cells were counted using a Leica epifluorescence stereomicroscope. Quantity

Cyanofluorescent cells were used to normalize the control and test sample by making a relative measurement of the quality of cells that received DNA efficiently for expression of transgenes.

[0131] When the excision cassette contained a FRT1 and FRT5 recombination site, it was expected that the incidence of cyanofluorescent cells that also express a yellow fluorescent protein would decrease to approximately less than 1% of the observed value, using an excision cassette containing two FRT1 recombination sites. These sites are therefore defined as non-recombinogenic.

B. Analysis based on luciferase enzymatic activity [0132] In the second analysis system, the equimolar mixture plasmids were re-used, simultaneously supplied by bombardment to the hi-II thyroid cells of immature embryos. For the purposes of this analysis, three plasmids are shown in Table 5.

Table 5

<td>FRT tested</td><td>Control</td>

<td>Actin :: FRT x: GUS: FRTx: FF-luciferase: nose *</td><td>Actin:: FRT x: FF-luciferase :: nose *</td>

<td>Nose :: Luciferase with ReniHa :: 35S term</td><td>Nose :: Luciferase from Renilla :: 35S term</td>

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

<td colspan="2">* FF = firefly luciferase; luciferase from ReniHa (Minko et af. (1999) Moth. Gen. Genet. 262: 421-425)</td>

[0133] Again, FRT1 was used to test the validity of the analysis system. In a follow-up study, actin constructs were introduced into thyroid cells: FRT1: FF-luciferase :: nos, Nos :: luciferase from Renilla :: 35S term and Ubi :: FLP :: pinll and after 2 days the tissue was extracted using the methods and solutions provided included in the set for the Promega Dual luciferase activity (Promega, Madison, Wi 53711). Multiple thyroid cells were individually extracted, and then the extracts were anaHz using fluoroscan for firefly luciferase activity followed by Renilla luciferase activity. As a result of using this mixture of constructs, it is expected that the expressed firefly luciferase protein produces 5,000 relative light units at 5,000 relative light units, and the protein from ReniHa luciferase produces about 15,000. When FRT1

PZ / 1656 / AR EP 1 907 553 B1 is used in a notch-activated cassette (Actin: FRT1: GUS: FRT1: firefly luci: 35s term), it is expected that firefly luciferase will produce about 4500 light units (about 90 % control). When FRT 5 is used in the excision cassette, fireworm luciferase activity is expected to decrease to approximately 670 light units (~ 15% of FRT1 value).

Reference example 7. Targeted insertion of the desired polynucleotide into corn

A. Determination of the target line [0134] To assess the FRT sequence for site-specific integration, a target was first created by stably integrating a polynucleotide containing a target having two functional FRT recombination sites, with the recombination sites being different and non-recombinogenic with respect to the second site.

This initial transformation is performed in Hi-II germplasm (or inbred lines) using standard maize transformation methods using Agrobacterium (see Example 5B). For example, to compare the relative efficiencies of FRT5 and FRT87 in a site-specific integration system, the following constructs were separately introduced into 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 [0135] Stable transformants were selected by visual evaluation of cells for yellow fluorescent callus tissue growing on glyphosate containing medium. Plants were regenerated and transferred to a greenhouse. Leaf samples were taken for Southern analysis. A single copy of the transgenic plant leaves were grown to maturity and crossed with wild type Hi-II (or inbred lines).

PZ / 1656 / AR EP 1 907 553 B1

Such transgenic products at this stage contained the FRT1-5 or FRT1-87 target site and were ready for site-specific recombinase mediated recombination.

B. Introduction of the transfer cassette by particle bombardment [0136] Immature embryos from a line having targets confirmed by yellow fluorescence expression were used for subsequent transformation. During the transformation process, transfer cassettes were introduced using standard particle bombardment methods (e.g., see Example 5A). In studies of progeny plants that contained integrated T-DNA with PHP20705 (FRT1-FRT87 target), the following insert containing the transfer cassette was used with a shotgun for populating the particles for retransformation:

PHP20915:

RB- CaMV35S Term / FRT1: bar :: pinll / Ubi: Ubi-intron: Renilla luciferase :: pinII / ln2-1 term: Am-Cyan1: FRT87 / CaMV35S Term -LB.

Immature embryos of progeny plants that contained integrated T-DNA with PHP20807 (FRT1-FRT5 target) were re-transformed using a molecular shotgun using the following plasmid:

PHP20954:

RB- CaMV35S Term / FRT1: bar :: pinII / Ubi: Ubi-intron: luciferase from Renilla :: pinII / ln2-1 term: Am-Cyan1: FRT5 / CaMV35S Term -LB.

[0137] For both plasmids containing the transfer cassette (PHP20915 and PHP20954), the bar and Cyan FP genes did not have a promoter. To reduce the likelihood that accidental inclusion would result in abnormal expression of any gene, the CaMV35S terminator FRT1 site was placed above. Each of these plasmids was transformed jointly into immature embryos from their respective target lines, together with plasmid PHP5096 (Ubi: Ubiintron :: FLPm :: pinII). PHP20915 or PHP20954 was mixed with the FLP containing plasmid (PHP5096), using up to 1 bombardment of 100 ng FRT containing plasmid and 10 ng FLP plasmid.

[0138] To prepare the delivered DNA, DNA solutions were added to a 50 μΙ gold stock solution (0.1 μg / μl of 0.6 micron gold particles). For example, 10 μl of a 0.1 μg / μl solution of PHP20915 or PHP20954 and 10 μl of a 0.01 μg / μl of solution PHP5096 are added to 30 μl of water initially. To this DNA mixture, 50 mL of gold stock solution was added and the mixture was briefly sonicated. Then, 5 μl TFX-50 (Promega Corp., 2800 Woods Hollow Road, Madison Wl 53711) was added and the mixture was placed on a rotary shaker at 100 rpm for 10 minutes. The mixture was briefly centrifuged to pellet the gold particles and the supernatant was removed. After removing the excess DNA / TFX solution, 120 μl absolute EtOH was added and 10 μl was suspended on macrocarriers typically used with the Dupont PDS-1000 helium gene shotgun. The gold particles and adherent DNA were allowed to dry on supports and then used for standard particle bombardment. After delivery of the plasmid containing the transfer cassette plus the plasmid containing FLP by re-transformation, immature embryos were placed on 560P medium for two weeks for recovery and then transferred to 3 mg / L bialaphos medium for selection. Effective recombination at both 5 '(FRT1) and 3' (FRT87 or 5) recombination targets will result in the activation of both genes, the bar gene and the cyanofluorescent protein gene when these structural genes are transferred into the functional vicinity of the Ubi promoter or Actin. Thus, specific cases of site-specific incorporation will be selected based on new, activated phenotypes. When callus tissue is large enough to take a sample, genomic DNA is extracted from the tissue and analyzed by PCR for the presence of products obtained by amplifying fragments using primers that include a new promoter-gene combination. Finally, leaf samples are taken from regenerated plants for Southern analysis to confirm proper recombination resulting in transfer of the donor cassette to the target genome locus.

After verifying the successful transformation of the loci, the plants were grown to maturity and crossed non-blooded or vegetatively propagated.

C. Introduction of the transfer cassette by crossing [0139] Transfer cassettes can be obtained by sexual crossing. In this example, stably transgenic cases are again used, containing a single copy of the T-DNA cassettes originally derived from Agrobacterium, containing PHP20705 or PHP20807. However, in this method

Stable transgenic donor cases are obtained using a vector selected from two Agrobacterium T-DNA vectors listed below.

1. A donor vector that complements PHP20705:

RB-Axig1 :: lEC1 :: pinII / Ubi Pro: Ubi-intron :: YFP :: pinII / -LB and

RB- ln2 :: FLP :: pinII- CaMV35S Term / FRT1: bar :: pinII / Ubi: Ubi-intron: Renilla luciferase :: pinII / ln2-1 term: Am-Cyan1: FRT87 / CaMV35S Term -LB

2. The donor vector complements PHP20807:

RB-Axig1 :: LEC1 :: pinII / Ubi Pro: Ubi-intron :: YFP :: pinII / -LB and

RB-ln2 :: FLP :: pinII- CaMV35S Term / FRT1: bar :: pinII / Ubi: Ubi-intron: Renilla luciferase :: pinII / ln2-1 term: Am-Cyan1: FRT5 / CaMV35S Term -LB [0140 ] For both of the above plasmids, the expression cassettes in the first T-DNA provide tools for selecting transgenic donor lines after transformation with Agrobacterium. The second T-DNA provides components for the intersecting cassette exchange. It should be noted that in both constructs, the inducible FLP expression cassette is located outside the FRT sites, and therefore, after successful exchange, does not transfer to the target site.

[0141] Recombination products having a transfer cassette are selected on the basis of visual selection of strongly growing yellow fluorescent callus tissue, regenerated, grown to maturity, and crossed to obtain donor seeds having a transfer cassette. Seeds from the target construct containing the T-DNA fragment from PHP20705, as well as seeds from the donor construct containing T-DNA from the above donor plasmid # 1 are planted and grown to maturity. During flowering, crossbreeding occurs between target and donor plants. The resulting seeds are planted and the plants are screened for a newly activated phenotype, which indicates successful recombination at two different FRT sites, in this case the activation of bialafos resistance is an indicator of proper recombination at the FRT5 site, and the activation of cyanofluorescence is

PZ / 1656 / AR EP 1 907 553 B1 as an indicator of proper recombination in FRT87. Similar crosses were obtained using target and transfer lines obtained from PHP20807 and donor plasmid # 2, respectively.

Example 8. Transformation of bacterial cells [0142] The new recombination sites disclosed herein can also be evaluated and used in bacterial cells such as E. coli. There are many known and readily available competent cell lines and bacterial plasmids. Isolated polynucleotides for transformation and transformation of bacterial cells can be obtained by any method known in the art. For example, transformation methods using E. coli and other bacterial cells, plasmid production, and phage use are described in detail, for example, in Current Protocols in Molecular Biology (FM Ausubei et al., (Ed.) (1994) collaborative work Greene Publishing Associates, Inc. and John Wiley & Sons,

Inc.). For example, the efficient electroporation protocol (Tung & Chow, Current Protocols in Molecular Biology, Appendix 32, Fall 1995) is summarized below.

[0143] Inoculate 100ml LB medium using 1ml overnight E. coli culture. incubation is carried out at 37 ° C with vigorous shaking until the culture reaches OD600 = 0.6. The culture is cooled on ice for 30 min, the cell pellet is separated by centrifugation at 4,000 xg for 15 min at 4 ° C: The cell pellet is washed twice with 50 ml of ice 10% glycerol. After the final washing, the cell pellet is suspended in a final volume of 0.2 ml in ice GYT medium (10% v / v glycerol; 0.125% w / v yeast extract; 0.25% w / v tritone). Electroporation is carried out in pre-chilled cuvettes using manufacturer-specified conditions, for example 0.5 ng plasmid DNA / transformation using the Gene Pulser kit (BioRad) at 25μF, 200 ohm, 2.5 kV. Immediately after electroporation, 1 ml of SOC medium is added and the cells are transferred to culture tubes. Incubation is carried out at 37 ° C for 1 h. A defined number of cells are spread on selective agar plates and incubated overnight at 37 ° C.

Example 9. Yeast transformation [0144] The new recombination sites disclosed herein can also be evaluated and used in yeast cells from which FLP recombinase and FRT sites were initially isolated. Commercially available and

Many strains of S. cerevisiae are published, plasmids used for transformation of these cells are available. PZ / 1656 / AR EP 1 907 553 B1 For example, strains are available from the American Clean Culture Collection (ATCC, Manassas, VA) and the Yeast Genetic Stock innovation center, which was transferred to ATCC in 1998. Other yeast lines such as S. pombe and P. pastoris, and the like are also available. For example, methods for yeast transformation, plasmid production, and the like are described in detail, for example, in Current Protocols in Molecular Biology (FM Ausubel et al. (Ed.) (1994), joint work of Greene Publishing Associates, Inc. and John Wiley & Sons,

Inc., see in particular Chapter 13). Yeast transformation methods include spheroplast transformation, electroporation and lithium acetate methods. The method of reversible, high-efficiency transformation of yeast is also widely described by Gietz & Woods ((2002) Methods Enzymol. 350: 87-96) transformation using lithium acetate, PEG 3500 and DNA carrier.

Example 10. Transformation of mammalian cells [0145] The new recombination sites disclosed herein can also be evaluated and used in mammalian cells such as CHO, HeLa, BALB / c, fibroblasts, embryonic mouse stem cells and the like. Many competent cell lines and plasmids on the market are known and are readily available, for example from ATCC (Manassas, VA). Isolated polynucleotides for transformation and transformation of mammalian cells can be obtained by any method known in the art. For example, methods for transforming mammalian cells and other eukaryotic cells, plasmid preparation, and the use of viruses are described in detail, for example, in Current Protocols in Molecular Biology (FM Ausubel et al. (Ed.) (1994) collaborative work Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., see in particular Chapter 9). For example, many methods are available, such as calcium phosphate transfection, electroporation, DEAE-dextran transfection, liposome transfection, microinjection, as well as viral techniques.

Reference example 11. In vitro recombinant cloning methods [0146] In Examples A, B and C below, the two starting nucleic acid molecules (eg, plasmids) are called "insertion donors" and "vectors

"The insertion donor contains a segment that will be attached to the new sequence provided by the donor vector. Recombination results in two sister molecules: the first is called the product (new clone desired) and the second is the by-product .

[0147] In the following examples, two pairs of plasmids were obtained to perform the in vitro recombinant cloning method in two different ways. One pair of plasmids obtained, Plasmid A and plasmid B, contains the FRT site and the lox site, for use with Cre and FLP recombinase. The second pair of plasmids, Plasmid D and Plasmid E, were constructed to contain the FRT (wild type) site for FLP and the second mutated FRT site (SEQ ID NO: 40), which differs from the wild type FRT site with 3 rules with a total of 30 base pairs. In this example, each plasmid contains a set of functional recombination sites, wherein the recombination sites are different and do not recombine with another site.

buffers:

[0148] Different buffers may be used in the reactions. For restriction enzymes, it is recommended to use buffers recommended by the manufacturer. Alternative buffers can easily be found in the literature or can be developed by a person skilled in the art. One example of buffers for lambda integrase contains 50 mM TrisHCl, pH 7.5-7.8, 70 mM KCI, 5 mM spermidine, 0.5 mM EDTA and 0.25 mg / ml bovine serum albumin, and optionally, 10% glycerol. An exemplary Cre P1 recombinase buffer contains 50 mM Tris-HCl pH 7.5, 33 mM NaCl, 5 mM spermidine and 0.5 mg / ml bovine serum albumin and an exemplary FLP buffer is discussed above in Example 2. An exemplary Cre and FLP recombinase buffer 50 mM Tfis-HCL at pH 7.5, 70 mM NaCl, 2mM MgCl2 and 0.1 mg / ml BSA, (Buchholz et al. (1996) Nucleic Acids Res. 24: 4256-4262). Buffers for other site-specific recombinases are known in the art or can be experimentally developed by those skilled in the art, especially in view of the buffers described above.

A. Recombinant cloning with FLP recombinase

[0149] Two plasmids were obtained. The donor plasmid (plasmid A) contains the following order: wild type FRT site, constitutive drug resistance marker (chloramphenicol resistance), origin of replication, constitutively expressed tet repressor protein gene (tetR), FRT site 5 and conditional drug resistance marker (kanamycin resistance when expressed under the control of the Tn10 transposon operator / promoter of the Tn10 transposon resistance). E. cells coli containing plasmid A show resistance to chloramphenicol at 30 μg / ml, but are sensitive to kanamycin at 100 μg / ml.

[0150] The donor insertion plasmid (plasmid B) contains in the following order: a wild type FRT site, another drug resistance marker (ampicillin resistance), a FRT site 5, an origin of replication and a multiple cloning site.

[0151] About 75 ng of each of plasmids A and B were mixed in a total volume of 30 µl FLP reaction buffer. Two 10 μl aliquots were transferred to new tubes. FLP protein was added to one tube. Both tubes were incubated at 37 ° C for 30 minutes and then at 70 ° C for 10 minutes. An aliquot of each reaction was diluted and transformed into DH5a. After expression, the samples were spread on plates containing 30 μg / ml chloramphenicol; 100 μg / ml ampicillin plus 200 μg / ml methicillin or 100 μg / ml kanamycin.

[0152] Colonies that showed chloramphenicol resistance, ampicillin resistance and kanamycin sensitivity after carrying out the recombination reaction contained a new vector product (plasmid C). Plasmid C contains the following order: wild type FRT site, constitutive drug resistance marker (chloramphenicol resistance), origin of replication, constitutively expressed tet repressor protein gene (tetR), FRT site 5 and ampicillin resistance marker.

[0153] To confirm the structure of the vector product (plasmid C), colonies exhibiting chloramphenicol resistance, ampicillin resistance and kanamycin sensitivity were collected and inoculated in medium containing 100 μg / ml kanamycin. Miniprep preparations were made, miniprep DNA was digested with appropriate restriction enzymes and electrophoretically analyzed. Plasmid C can be identified based on the expected size of the plasmid product and the fragments obtained by restriction enzyme digestion.

B. Recombinant cloning with FLP recombinase and Cre recombinase

[0154] The plasmids used in the present method are analogous to those described above, except that Plasmid D, the plasmid donor vector, contains a loxP site instead of the wild type FRT site and Plasmid E, donor plasmid insertional, contains a loxP site instead of a wild type FRT site.

[0155] About 500 ng of plasmid E and plasmid D were precipitated with ethanol and resuspended in 40 μl Cre / FLP reaction buffer (described above). The reaction mixtures were incubated at 37 ° C for 30 minutes and then at 70 ° C for 10 minutes. TE buffer (90 µL; TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) was added to each reaction and 1 μl of each reaction was transformed into E. coli DH5a. Transformation mixtures were spread on plates containing 100 pg / ml ampicillin plus 200 pg / ml methicillin; 30 pg / ml chloramphenicol or 100 pg / ml kanamycin.

[0156] Colonies that showed chloramphenicol resistance, ampicillin resistance and kanamycin sensitivity after carrying out the recombination reaction contained a new vector product (plasmid F). Plasmid F contains in the following order: wild type loxP site, constitutive drug resistance marker (chloramphenicol resistance), origin of replication, constitutively expressed tet repressor protein gene (tetR), FRT site 5 and ampicillin resistance marker.

[0157] To confirm the structure of the vector product (plasmid F), colonies exhibiting chloramphenicol resistance, ampicillin resistance and kanamycin sensitivity were collected and inoculated in medium containing 900 pg / ml kanamycin. Miniprep preparations were made, miniprep DNA was digested with appropriate restriction enzymes and electrophoretically analyzed. Plasmid F can be identified based on the expected size of the plasmid product and the fragments obtained by restriction enzyme digestion.

C. In vitro recombinant cloning into the subclone of the chloramphenicol acetyltransferase gene using eukaryotic cell expression vector [0158] The donor insertion plasmid, Plasmid G, containing in the following order: wild type FRT site, promoter lacking the chloramphenicol acetyltransferase gene from E. coli, site FRT 5, origin of replication and a constitutive drug resistance marker (ampicillin resistance).

[0159] A plasmid donor vector, Plasmid H, was obtained, containing in the following order: kanamycin resistance gene, origin of replication, eukaryotic cytomegalovirus promoter, wild type FRT site, constitutively expressed tet repressor protein gene (tetR), chloramphenicol resistance gene and site FRT 5. One microliter of each plasmid, typically about 50 ng raw

Miniprep DNA, was combined with a 10μ1 reaction mixture containing the FLP reaction buffer and FLP recombinase. PZ / 1656 / AR EP 1 907 553 B1 After incubation at 30 ° C for 30 minutes and at 75 ° C for 10 minutes, one microliter was transformed into the competent E. coli DH5a strain (Life Technologies, Inc.). Transformants were spread on agar plates containing 200 μg / ml kanamycin and incubated at 37 ° C overnight. In contrast, the control reaction was identical, but contained only the donor plasmid vector.

[0160] To confirm the structure of the vector product (plasmid I), "miniprep" preparations were made, miniprep DNA was digested with appropriate restriction enzymes and electrophoretically analyzed. Plasmid I can be identified based on the expected size of the plasmid product and to obtain confirmation of the presence of chloramphenicol acetyltransferase, the fragments obtained by restriction enzyme digestion were cloned below the CMV promoter.

[0161] The articles used refer to one or more (i.e., at least one) grammatical subject. For example, "element" means one or more elements.

PZ / 1656 / AR EP 1 907 553 B1

SEQUENCE LIST [0162] <110> Pioneer Hi-Bred International, Inc.

<120> New FRT recombination sites and methods of their use <130> 1525-PCT <150> 60 / 700.225 <151> 2005-07-18 <160> 72 <170> FastSEQ for Windows version 4.0 <210> 1 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 12 <400> 1 tctatgta 8 <210> 2 <211> 8 <212> DNA

<213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 57 <400> 2 ttttctaa 8 <210> 3 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 85 <400> 3 tttcttga 8 <210> 4 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 87 <400> 4 tttctgga 8 <210> 5

PZ / 1656 / AR EP 1 907 553 B1 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 53 site <400> 5 tgtaaaaa 8 <210> 6 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 62 <400> 6 tttaggta 8 <210> 7 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 78 <400> 7

Pga / 1656 / AR EP 1 907 553 B1 tgaaaaga 8 <210> 8 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 34 <400> 8 tgtaatga 8 <210> 9 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT 70 FRT 70 linker sequence <400> 9 tatacaaa 8 <210> 10 <211> 8 <212> DNA <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> New FRT linker sequence of FRT 76 <400> 10 ttccataa 8 <210> 11 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 89 <400> 11 tctctaga 8 <210> 12 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker FRT 43 <400> 12 ttccgaga 8 <210> 13 <211> 8

PZ / 1656 / AR EP 1 907 553 B1 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 45 <400> 13 tctcttga 8 <210> 14 <211> 8 <212> DNA <213>. Artificial sequence <220>

<223> New FRT linker sequence of FRT 55 site <400> 14 tccacaga 8 <210> 15 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 65 <400> 15 tgattgga

PZ / 1656 / AR EP 1 907 553 B1 <210> 16 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT site <400> 16 ttttgtga 8 <210> 17 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 74 <400> 17 tgagagaa 8 <210> 18 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> New FRT linker sequence of FRT 86 site

PZ / 1656 / AR EP 1 907 553 B1 <400> 18 tttctcga 8 <210> 19 <211> 11 <212> DNA <213> Artificial sequence <220>

<223> 5 'FLP binding site (symmetry element) of the wild type FRT <400> 19 agttcctata c 11 <210> 20 <211> 11 <212> DNA <213> Artificial sequence <220>

<223> 3 'FLP binding site (symmetry element) FRT wild site <400> 20 gaataggaac t 11 <210> 21 <211> 30 <212> DNA <213> Artificial sequence

PZ / 1656 / AR <220>

<223> New minimal FRT 12 site <400> 21 agttcctata ctctatgtag aataggaact 30 <210> 22 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site 57 <400> 22 agttcctata cttttctaag aataggaact 30 <210> 23 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 85 site <400> 23 agttcctata ctttcttgag aataggaact 30 <210> 24

EP 1 907 553 B1 <211> 30

PZ / 1656 / AR EP 1 907 553 B1 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 87 site <400> 24 agttcctata ctttctggag aataggaact 30 <210> 25 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 53 site <400> 25 agttcctata ctgtaaaaag aataggaact 30 <210> 26 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimum FRT 62 site <400> 26 agttcctata ctttaggtag aataggaact

PZ / 1656 / AR EP 1 907 553 B1 <210> 27 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site 78 <400> 27 agttcctata ctgaaaagag aataggaact 30 <210> 28 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 34 site <400> 28 agttcctata ctgtaatgag aataggaact 30 <210> 29 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimum FRT 70 space

PZ / 1656 / AR <400> 29 agttcctata ctatacaaag aataggaact <210> 30 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site <400> 30 agttcctata cttccataag aataggaact <210> 31 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site <400> 31 agttcctata ctctctagag aataggaact <210> 32 <211> 30 <212> DNA

EP 1 907 553 B1 <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> New minimal FRT site 43 <400> 32 agttcctata cttccgagag aataggaact 30 <210> 33 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 45 site <400> 33 agttcctata ctctcttgag aataggaact 30 <210> 34 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT space 55 <400> 34 agttcctata ctccacagag aataggaact 30 <210> 35 <211> 30

PZ / 1656 / AR EP 1 907 553 B1 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT 65 site <400> 35 agttcctata ctgattggag aataggaact 30 <210> 36 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site 69 <400> 36 agttcctata cttttgtgag aataggaact 30 <210> 37 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimum FRT 74 site <400> 37 agttcctata ctgagagaag aataggaact

PZ / 1656 / AR EP 1 907 553 B1 <210> 38 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> New minimal FRT site 86 <400> 38 agttcctata ctttctcgag aataggaact 30 <210> 39 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimal wild-type site FRT <400> 39 agttcctata ctttctagag aataggaact .30 <210> 40 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimal FRT5 mutated site

PZ / 1656 / AR EP 1 907 553 B1 <400> 40 agttcctata ctcttttgag aataggaact 30 <210> 41 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimal FRT6 mutated site <400> 41 agttcctata ctttttgaag aataggaact 30 <210> 42 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimal FRT7 mutated site <400> 42 agttcctata cttattgaag aataggaact 30 <210> 43 <211> 8 <212> DNA <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> Linker sequence of wild type FRT recombination site <400> 43 tttctaga 8 <210> 44 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT 5 recombination site linker sequence <400> 44 cttttgaa 8 <210> 45 <211> 1032 <212> DNA <213> Bacteriophage C1 <220>

<221> CDS <222> (1) ... (1016) <400> 45

PZ / 1656 / AR EP 1 907 553 B1

<td>atg Underworld 1</td><td colspan="2">tcc aat Cheese Asn</td><td>tta Leu</td><td>cOT Leu 5</td><td>acc Thr</td><td>gta val</td><td>cac His</td><td>caa Gln</td><td>aat own 10</td><td>TTG Leu</td><td>Cct Pro</td><td colspan="2">gca tta Ala Leu</td><td>ccg Pro 15</td><td>gtc val</td><td> 48</td>

<td>Asp</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Asp</td><td>Glu</td><td>val</td><td>Arg</td><td>lys</td><td>own</td><td>Leu</td><td>Underworld</td><td>Asp</td><td>Underworld</td><td>phe</td><td>Arg</td><td></td>

<td>Asp</td><td>Arg</td><td>Gln</td><td>ala</td><td>phe</td><td>Cheese</td><td>Glu</td><td>His</td><td>Thr</td><td>Trp</td><td>lys</td><td>Underworld</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>val</td><td></td>

<td>Cys</td><td>Arg</td><td>Cheese</td><td>Trp</td><td>ala</td><td>ala</td><td>Trp</td><td>Cys</td><td>lys</td><td>Leu</td><td>own</td><td>own</td><td>Arg</td><td>lys</td><td>Trp</td><td>phe</td><td></td>

<td>Underworld</td><td>Leu</td><td>His</td><td>Arg</td><td>Arg</td><td>Cheese</td><td>Gly</td><td>Leu</td><td>Pro</td><td>Arg</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>Cheese</td><td>own</td><td>ala</td><td></td>

<td>val</td><td>Cheese</td><td>Leu</td><td>val</td><td>Underworld</td><td>Arg</td><td>Arg</td><td>How much</td><td>Arg</td><td>lys</td><td>Glu</td><td>own</td><td>val</td><td>Asp</td><td>ala</td><td>Gly</td><td></td>

PZ / 1656 / AR EP 1 907 553 B1

<td rowspan="2">gtt val 145</td><td rowspan="2">cgt Arg</td><td colspan="2">tca ctc</td><td rowspan="2">atg Underworld</td><td rowspan="2">gaa Glu 150</td><td rowspan="2">aat own</td><td rowspan="2">agc Cheese</td><td rowspan="2">gat Asp</td><td rowspan="2">cgc Arg</td><td colspan="2">tgc cag</td><td rowspan="2">gat Asp</td><td rowspan="2">ata How much</td><td rowspan="2">cgt Arg</td><td rowspan="2">aat own 160</td><td rowspan="2"> 480</td>

<td>Cheese</td><td>Leu</td><td>Cys 155</td><td>Gin</td>

<td>How much</td><td>ala</td><td>Arg</td><td>How much 180</td><td>Arg</td><td>val</td><td>lys</td><td>Asp</td><td>How much 185</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Asp</td><td>Gly 190</td><td>Gly</td><td>Arg</td><td></td>

<td>Underworld</td><td>Leu</td><td>How much 195</td><td>His</td><td>How much</td><td>Gly</td><td>Arg</td><td>Thr 200</td><td>lys</td><td>Thr</td><td>Leu</td><td>val</td><td>Cheese 205</td><td>Thr</td><td>ala</td><td>Gly</td><td></td>

<td>val</td><td>Glu 210</td><td>lys</td><td>ala</td><td>Leu</td><td>Cheese</td><td>Leu 215</td><td>Gly</td><td>val</td><td>Thr</td><td>lys</td><td>Leu 220</td><td>val</td><td>Glu</td><td>Arg</td><td>Trp</td><td></td>

<td>att</td><td>tcc</td><td>gtc</td><td>tct</td><td>GGT</td><td>gta</td><td>GCT</td><td>gat</td><td>gat</td><td>ccg</td><td>aat</td><td>aac</td><td>trays</td><td>cOT</td><td>ttt</td><td>TGC</td><td> 720</td>

<td>How much 225</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Gly</td><td>val 230</td><td>ala</td><td>Asp</td><td>Asp</td><td>Pro</td><td>own 235</td><td>own</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Cys 240</td><td></td>

<td>Arg</td><td>val</td><td>Arg</td><td>lys</td><td>own 245</td><td>Gly</td><td>val</td><td>ala</td><td>ala</td><td>Pro 250</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Gin 255</td><td>Leu</td><td></td>

<td>Cheese</td><td>Thr</td><td>Arg</td><td>ala 260</td><td>Leu</td><td>Glu</td><td>Gly</td><td>How much</td><td>phe 265</td><td>Glu</td><td>ala</td><td>Thr</td><td>His</td><td>Arg 270</td><td>Leu</td><td>How much</td><td></td>

<td>trays</td><td>GGC</td><td>GCT</td><td>aag</td><td>gat</td><td>gac</td><td>tct</td><td>GGT</td><td>cag</td><td>aga</td><td>trays</td><td>cOT</td><td>gcc</td><td>TGG</td><td>tct</td><td>GGA</td><td> 8 64</td>

<td>Tyr</td><td>Gly</td><td>ala 275</td><td>lys</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>Gly 280</td><td>Gin</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>ala 285</td><td>Trp</td><td>Cheese</td><td>Gly</td><td></td>

<td>His</td><td>Cheese 290</td><td>ala</td><td>Arg</td><td>val</td><td>Gly</td><td>ala 295</td><td>ala</td><td>Arg</td><td>Asp</td><td>Underworld</td><td>ala 300</td><td>Arg</td><td>ala</td><td>Gly</td><td>val</td><td></td>

<td>Cheese 305</td><td>How much</td><td>Pro</td><td>Glu</td><td>How much</td><td>Underworld 310</td><td>Gin</td><td>ala</td><td>Gly</td><td>Gly</td><td>Trp 315</td><td>Thr</td><td>own</td><td>val</td><td>own</td><td>How much 320</td><td></td>

<td>val</td><td>Underworld</td><td>own</td><td>Tyr</td><td>How much 325</td><td>Arg</td><td>own</td><td>Leu</td><td>Asp</td><td>Cheese 330</td><td>Glu</td><td>Thr</td><td>Gly</td><td>ala</td><td>Underworld 335</td><td>val</td><td></td>

cgc ctg ct ggaagatggc gattag 1032

Arg Leu <210> 46 <211> 338 <212> PRT <213> Bacteriophage C1 <400> 46

PZ / 1656 / AR EP 1 907 553 B1

Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val

<td>Cys</td><td>Arg</td><td>Cheese</td><td>Trp</td><td>ala</td><td>ala</td><td>Trp</td><td>Cys</td><td>lys</td><td>Leu</td><td>own</td><td>own</td><td>Arg</td><td>lys</td><td>Trp</td><td>phe</td>

55 60

Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala

<td>val</td><td>Arg</td><td>Cheese</td><td>Leu</td><td>Underworld</td><td>Glu</td><td>own</td><td>Cheese</td><td>Asp</td><td>Arg</td><td>Cys</td><td>Gln</td><td>Asp</td><td>How much</td><td>Arg</td><td>own</td>

<td>How much</td><td>ala</td><td>Arg</td><td>How much</td><td>Arg</td><td>val</td><td>lys</td><td>Asp</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Asp</td><td>Gly</td><td>Gly</td><td>Arg</td>

<td>Underworld</td><td>Leu</td><td>How much</td><td>His</td><td>How much</td><td>Gly</td><td>Arg</td><td>Thr</td><td>lys</td><td>Thr</td><td>Leu</td><td>val</td><td>Cheese</td><td>Thr</td><td>ala</td><td>Gly</td>

<td>val</td><td>Glu</td><td>lys</td><td>ala</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>val</td><td>Thr</td><td>lys</td><td>Leu</td><td>val</td><td>Glu</td><td>Arg</td><td>Trp</td>

210 215 . 220

Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys

<td>Arg</td><td>val</td><td>Arg</td><td>lys</td><td>own</td><td>Gly</td><td>val</td><td>ala</td><td>ala</td><td>Pro</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Cheese</td><td>Gln</td><td>Leu</td>

<td>Cheese</td><td>Thr</td><td>Arg</td><td>ala</td><td>Leu</td><td>Glu</td><td>Gly</td><td>How much</td><td>phe</td><td>Glu</td><td>ala</td><td>Thr</td><td>His</td><td>Arg</td><td>Leu</td><td>How much</td>

<td>Tyr</td><td>Gly</td><td>ala</td><td>lys</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>Gly</td><td>Gln</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Cheese</td><td>Gly</td>

<td>His</td><td>Cheese</td><td>ala</td><td>Arg</td><td>val</td><td>Gly</td><td>ala</td><td>ala</td><td>Arg</td><td>Asp</td><td>Underworld</td><td>ala</td><td>Arg</td><td>ala</td><td>Gly</td><td>val</td>

<td>Cheese</td><td>How much</td><td>Pro</td><td>Glu</td><td>How much</td><td>Underworld</td><td>Gln</td><td>ala</td><td>Gly</td><td>Gly</td><td>Trp</td><td>Thr</td><td>own</td><td>val</td><td>own</td><td>How much</td>

<td>val</td><td>Underworld</td><td>own</td><td>Tyr</td><td>How much</td><td>Arg</td><td>own</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Glu</td><td>Thr</td><td>Gly</td><td>ala</td><td>Underworld</td><td>val</td>

<210> 47 <211> 1032 <212> DNA <213> Artificial sequence <220>

<223> Nucleotide sequence coding for a Cre protein from bacteriophage P1 having preferred corn-derived codons (moCRE) <221> CDS

PZ / 1656 / AR <222> (1) ... (1032) <400> 47

EP 1 907 553 B1 atg tcc aac ctg ctc acg gtt cac cag aac ctt ccg gct ctt cca gtg 48

Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val

VP / 1656 / AR

EP 1 907 553 B1

<td>Asp</td><td>ala</td><td>Thr</td><td>Cheese twenty</td><td>Asp</td><td>Glu</td>

<td>Asp</td><td>Arg</td><td>Gln 35</td><td>ala</td><td>phe</td><td>Cheese</td>

<td>Cys</td><td>Arg 50</td><td>Cheese</td><td>Trp</td><td>ala</td><td>ala</td>

<td>Underworld</td><td>Leu</td><td>His</td><td>Arg 100</td><td>Arg</td><td>Cheese</td>

<td>val</td><td>Cheese</td><td>Leu 115</td><td>val</td><td>Underworld</td><td>Arg</td>

<td>val 145</td><td>Arg</td><td>Cheese</td><td>Leu</td><td>Underworld</td><td>Glu 150</td>

<td>Underworld</td><td>Leu</td><td>How much 195</td><td>His</td><td>How much</td><td>Gly</td>

<td>val</td><td>Glu 210</td><td>lys</td><td>ala</td><td>Leu</td><td>Cheese</td>

<td>How much 225</td><td>Cheese</td><td>val</td><td>Cheese</td><td>Gly</td><td>val 230</td>

gtc agg aag aac ctc atg Val Arg Lys Asn Leu Met gag cac acc tgg aag atg Glu His Thr Trp Lys Met tgg tgc aag ctg aac aac Trp Cys Lys Leu Asn Asn

60 gtg agg gat tac ctt ctg Val Arg Asp Tyr Leu Leu acc atc cag caa cac ctt Thr Ile Gln Gln His Leu ggc ctc ccg cgc ccc agc Gly Leu Pro Arg Pro Ser

105 cgc atc agg aag gaa aac Arg Ile Arg Lys Glu Asn

120 ctc gcg ttc gag agg acc Leu Ala Phe Glu Arg Thr 135 140 aac agc gac agg tgc cag Asn Ser Asp Arg Cys Gln

155 gca tac aac acg ctc ctc Ala tyr Asn Thr Leu Leu

170 aag gac att agc cgc acc Lys Asp Ile Ser Arg Thr

185 agg acc aag acg ctc gtt Arg Thr Lys Thr Leu Val

200 ctc gga gtg acc aag ctc Leu Gly Val Thr Lys Leu 215 220 gcg gac gac cca aac aac Ala Asp Asp Pro Asn Asn

235 gtg gct gcc cct agc gcc Val Ala Ala Pro Ser Ala

250

<td>Asp</td><td>Underworld thirty</td><td>phe</td><td>Arg</td><td></td>

<td>Leu 45</td><td>Leu</td><td>Cheese</td><td>val</td><td></td>

<td>trays</td><td>cOT</td><td>caa</td><td>GCT</td><td> 240</td>

<td>Asp</td><td>Cheese 110</td><td>own</td><td>ala</td><td></td>

<td>Cheese 205</td><td>Thr</td><td>ala</td><td>Gly</td><td></td>

<td>trays</td><td>ctc</td><td>ttc</td><td>TGC</td><td> 720</td>

<td>Thr</td><td>Cheese</td><td>Gln 255</td><td>Leu</td><td></td>

PZ / 1656 / AR EP 1 907 553 B1

<td rowspan="2">agc Cheese</td><td rowspan="2">ACG Thr</td><td colspan="2">agg gcc</td><td rowspan="2">TTG Leu</td><td rowspan="2">gaa Glu</td><td rowspan="2">GGT Gly</td><td rowspan="2">att How much</td><td colspan="2">ttc gag</td><td colspan="2">gcc acc</td><td rowspan="2">cac His</td><td rowspan="2">cgc Arg 270</td><td rowspan="2">cOT Leu</td><td rowspan="2">atc How much</td><td rowspan="2"> 816</td>

<td>trays</td><td>GGC</td><td>GCG</td><td>aag</td><td>gat</td><td>gac</td><td>agc</td><td>GGT</td><td>caa</td><td>cgc</td><td>trays</td><td>ctc</td><td>GCA</td><td>TGG</td><td>tcc</td><td>ggg</td><td> 864</td>

<td>Tyr</td><td>Gly</td><td>ala</td><td>lys</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>Gly</td><td>Gln</td><td>Arg</td><td>Tyr</td><td>Leu</td><td>ala</td><td>Trp</td><td>Cheese</td><td>Gly</td><td></td>

<td>Cheese</td><td>How much</td><td>Pro</td><td>Glu.</td><td>How much</td><td>Underworld</td><td>Gln</td><td>ala</td><td>Gly</td><td>Gly</td><td>Trp</td><td>Thr</td><td>own</td><td>val</td><td>own</td><td>How much</td><td></td>

<td>gtc</td><td>atg</td><td>aac</td><td>trays</td><td>att</td><td>cgc</td><td>aac</td><td>ctt</td><td>gac</td><td>agc</td><td>gag</td><td>ACG</td><td>GGC</td><td>GCA</td><td>atg</td><td>gtt</td><td> 1008</td>

340 <210> 48 <211> 1260 <212> DNA <213> Saccharomyces cerevisiae <220>

<221> CDS <222> (1) ... (1260) <400> 48

VP / 1656 / AR

EP 1 907 553 B1 atg cca caa ttt ggt ata tta tgt aaa aca cca cct aag gtg ctt gtt Met Pro Gln Phe Gly Ile Leu Cys Lys Thr Pro Pro Lys Val Leu Val cgt cag ttt gtg gaa agg ttt gaa aga cct tca ggt gag aaa ata gca Arg Gln Phe Val Glu Arg Phe Glu Arg Pro Ser Gly Glu Lys Ile Ala tta tgt gct gct cct acc tat tta tgt tgg atg att aca cat aac Leu Cys Ala Ala Glu Leu Thr Tyr Leu Cys Trp Met Ile Thr His own

144 gga aca gca atc aag aga gcc aca ttc atg agc tat aat act atc ata Gly Thr Ala Ile Lys Arg Ala Thr Phe Met Ser Tyr Asn Thr Ile Ile

192 agc aat tcg ctg agt ttc gat att gtc aat aaa tca ctc cag ttt aaa Cheese Asn Cheese Leu Cheese Phe Asp Ile Val Asn Lys Cheese Leu Gln Phe · Lys

240

7.0 tac aag acg caa aaa gca aca att ctg gaa gcc tca tta aag aaa ttg Tyr Lys Thr Gln Lys Ala Thr Ile Leu Glu Ala Ser Leu Lys Lys Leu

288 att cct gct tgg gaa ttt aca att att cct tac tat gga caa aaa cat

336

VP / 1656 / AR

Ile Pro Ala Trp Glu Phe Thr Ile Ile 100 105 caa tct gat atc act gat att gta agt Gln Ser Asp Ile Thr Asp Ile Val Ser

115 120 tca tcg gaa gaa gca gat aag gga aat

Cheese Cheese Glu Glu Ala Asp Lys Gly Asn

130 135 aaa gca ctt eta agt gag ggt gaa age

Lys Ala Leu Leu Cheese Glu Gly Glu Cheese

145 150 ata eta aat tcg ttt gag tat act tcg

Ile Leu Asn Ser Phe Glu Tyr Thr Ser

165 tta tac caa ttc ctc ttc eta gct act

Leu Tyr Gln Phe Leu Phe Leu Ala Thr

180 185 age gat att aag aac gtt gat ccg aaa

Asp Asp Lys Asn v'al Asp Pro Lys cheese

195 200 aag tat ctg gga gta ata atc cag tgt

Lys Tyr Leu Gly Val Ile Ile Gln Cys

210 215 age gtt agt agg cac ata tac ttc ttt

Ser Val Ser Arg His Ile Tyr Phe Phe

225 . 230 cca ctt gta tat ttg gat gaa ttt ttg

Pro Leu Val Tyr Leu Asp Glu Phe Leu

245 aaa ega gta aat agg acc ggc aat tct

Lys Arg Val Asn Arg Thr Gly · Asn Ser

260 265 caa tta tta aaa gat aac tta gtc aga

Gln Leu Leu Lys Asp Asn Leu Val Arg

275 280 aaa aat gcg cct tat tca atc ttt gct

Lys Asn Ala Pro Tyr Cheese Ile Phe Ala

290 295 cac att gga aga cat ttg atg acc tca

His Ile Gly Arg His Leu Met Thr Ser

305 310 acg gag ttg act aat gtt gtg gga aat

Thr Glu Leu Thr Asn Val Val Gly Asn • 325 gee gtg gee agg aca acg tat act cat

Ala Val Ala Arg Thr Thr Tyr Thr His

EP 1 907 553 B1

Pro Tyr Tyr Gly Gln Lys His 110 agt ttg caa tta cag ttc gaa 384

Cheese Leu Gln Leu Gln Phe Glu 125 age cac agt aaa aaa atg ctt 432

Ser His Ser Lys Lys Met Leu 140 atc tgg gag atc act gag aaa 480

Ile Trp Glu Ile Thr Glu Lys

155 160 aga ttt aca aaa aca aaa act 528

Arg Phe Thr Lys Thr Lys Thr

170 175 ttc atc aat tgt gga aga ttc 576

Phe Ile Asn Cys Gly Arg Phe '190 tca ttt aaa tta gtc caa aat 624 _ ni__τ. . _ v___ i Ί - -n __ otii triiti jjyo Liuii vct ± kjj.ii zisu

205 tta gtg aca gag aca aag aca 672

Leu Val Thr Glu Thr Lys Thr

220 age gca agg ggt agg atc gat 720

Cheese Ala Arg Gly Arg Ile Asp 235 240 agg aat tct gaa cca gtc eta 768

Arg Asn Ser Glu Pro Val Leu

250 255 tca age aat aaa cag gaa tac 816

Cheese Cheese Asn Lys Gln Glu Tyr 270 tcg tac aat aaa gct ttg aag 864

Cheese Tyr Asn Lys Ala Leu Lys 285 ata aaa aat ggc cca aaa tct 912

Ile Lys Asn Gly Pro Lys Ser 300 ttt ctt tca atg aag ggc eta 960

Phe Leu Ser Met Lys Gly Leu 315 320 tgg age gat aag cgt gct tct 1008

Trp Cheese Asp Lys Arg Ala Cheese

330 335 cag ata aca gca ata cct gat 1056

Gln Ile Thr Ala Ile Pro Asp

EP 1 907 553 B1

VP / 1656 / AR

340 345 cac tac ttc gca cta gtt tct cgg tac

His Tyr Phe Ala Leu Val Ser Arg Tyr

355 360 aag gaa atg ata gca ttg aag gat gag

Lys Glu Met Ile Ala Leu Lys Glu

370 375 cag cat ata gaa cag cta. aag ggt agt

Gln His Ile Glu Gln Leu Lys Gly Ser

385 390 ccc gca tgg aat ggg ata ata tca cag

Pro Ala Trp Asn Gly Ile Ile Ser Gln

405 tcc tac ata aat.

Tyr Ile Asn. Cheese

420

<td>Tyr</td><td>ala</td><td>Tyr</td><td>Asp 365</td><td>Pro</td><td>How much</td><td>Cheese</td><td></td>

<td>GCT</td><td>gaa</td><td>GGA</td><td>agc</td><td>ata</td><td>ega</td><td>trays</td><td> 1200</td>

<td>ala</td><td>Glu 395</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Arg</td><td>Tyr 400</td><td></td>

<td>gag</td><td>gta</td><td>cta</td><td>gac</td><td>trays</td><td>ctt</td><td>tCA</td><td> 1248</td>

<td>Glu 410</td><td>val</td><td>Leu</td><td>Asp</td><td>Tyr</td><td>Leu 415</td><td>Cheese</td><td></td>

1260 <210> 49 <211> 420 <212> PRT <213> Saccharomyces cerevisiae <400> 49

PZ / 1656 / AR EP 1 907 553 B1

<td>Underworld</td><td>Pro</td><td>Gln</td><td>phe</td><td>Gly</td><td>How much</td><td>Leu</td><td>Cys</td><td>lys</td><td>Thr</td><td>Pro</td><td>Pro</td><td>lys</td><td>val</td><td>Leu</td><td>val</td>

<td>Arg</td><td>Gln</td><td>phe</td><td>val</td><td>Glu</td><td>Arg</td><td>phe</td><td>Glu</td><td>Arg</td><td>Pro</td><td>Cheese</td><td>Gly</td><td>Glu</td><td>lys</td><td>How much</td><td>ala</td>

<td>Leu</td><td>Cys</td><td>ala</td><td>ala</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Tyr</td><td>Leu</td><td>Cys</td><td>Trp</td><td>Underworld</td><td>How much</td><td>Thr</td><td>His</td><td>own</td>

<td>Gly</td><td>Thr</td><td>ala</td><td>How much</td><td>lys</td><td>Arg</td><td>ala</td><td>Thr</td><td>phe</td><td>Underworld</td><td>Cheese</td><td>Tyr</td><td>own</td><td>Thr</td><td>How much</td><td>How much</td>

<td>Cheese</td><td>own</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>phe</td><td>Asp</td><td>How much</td><td>val</td><td>own</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Gln</td><td>phe</td><td>lys</td>

<td>Tyr</td><td>lys</td><td>Thr</td><td>Gln</td><td>lys</td><td>ala</td><td>Thr</td><td>How much</td><td>Leu</td><td>Glu</td><td>ala</td><td>Cheese</td><td>Leu</td><td>lys</td><td>'Lys</td><td>Leu</td>

<td>Gln</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Thr</td><td>Asp</td><td>How much</td><td>val</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gln</td><td>Leu</td><td>Gln</td><td>phe</td><td>Glu</td>

<td>Cheese</td><td>Cheese</td><td>Glu</td><td>Glu</td><td>ala</td><td>Asp</td><td>lys</td><td>Gly</td><td>own</td><td>Cheese</td><td>His</td><td>Cheese</td><td>lys</td><td>lys</td><td>Underworld</td><td>Leu</td>

<td>lys</td><td>ala</td><td>Leu</td><td>Leu</td><td>Cheese</td><td>Glu</td><td>Gly</td><td>Glu</td><td>Cheese</td><td>How much</td><td>Trp</td><td>Glu</td><td>How much</td><td>Thr</td><td>Glu</td><td>lys</td>

<td>How much</td><td>Leu</td><td>own</td><td>Cheese</td><td>phe</td><td>Glu</td><td>Tyr</td><td>Thr</td><td>Cheese</td><td>Arg</td><td>phe</td><td>Thr</td><td>lys</td><td>Thr</td><td>lys</td><td>Thr</td>

<td>Cheese</td><td>Asp</td><td>How much</td><td>lys</td><td>own</td><td>val</td><td>Asp</td><td>Pro</td><td>lys</td><td>Cheese</td><td>phe</td><td>lys</td><td>Leu</td><td>val</td><td>Gln</td><td>own</td>

<td>Cheese</td><td>val</td><td>Cheese</td><td>Arg</td><td>His</td><td>How much</td><td>Tyr</td><td>phe</td><td>phe</td><td>Cheese</td><td>ala</td><td>Arg</td><td>Gly</td><td>Arg</td><td>How much</td><td>Asp</td>

<td>Pro</td><td>Leu</td><td>val</td><td>Tyr</td><td>Leu</td><td>Asp</td><td>Glu</td><td>phe</td><td>Leu</td><td>Arg</td><td>own</td><td>Cheese</td><td>Glu</td><td>Pro</td><td>val</td><td>Leu</td>

<td colspan="4">Lys Arg Val Asn</td><td rowspan="2">Arg</td><td rowspan="2">Thr</td><td colspan="2" rowspan="2">Gly Asn</td><td rowspan="2">Cheese 265</td><td colspan="4" rowspan="2">Cheese Cheese Asn Lys</td><td rowspan="2">Gln 270</td><td rowspan="2">Glu</td><td rowspan="2">Tyr</td>

<td>Gln</td><td>Leu</td><td>Leu</td><td>lys</td><td>Asp</td><td>own</td><td>Leu</td><td>val</td><td>Arg</td><td>Cheese</td><td>Tyr</td><td>own</td><td>lys</td><td>ala</td><td>Leu</td><td>lys</td>

<td>lys</td><td>own</td><td>ala</td><td>Pro</td><td>Tyr</td><td>Cheese</td><td>How much</td><td>phe</td><td>ala</td><td>How much</td><td>lys</td><td>own</td><td>Gly</td><td>Pro</td><td>lys</td><td>Cheese</td>

<td>His</td><td>How much</td><td>Gly</td><td>Arg</td><td>His</td><td>Leu</td><td>Underworld</td><td>Thr</td><td>Cheese</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Gly</td><td>Leu</td>

<td>Thr</td><td>Glu</td><td>Leu</td><td>Thr</td><td>own</td><td>val</td><td>val</td><td>Gly</td><td>own</td><td>Trp</td><td>Cheese</td><td>Asp</td><td>lys</td><td>Arg</td><td>ala</td><td>Cheese</td>

<td>His</td><td>Tyr</td><td>phe</td><td>ala</td><td>Leu</td><td>val</td><td>Cheese</td><td>Arg</td><td>Tyr</td><td>Tyr</td><td>ala</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>How much</td><td>Cheese</td>

<td>lys</td><td>Glu</td><td>Underworld</td><td>How much</td><td>ala</td><td>Leu</td><td>lys</td><td>Asp</td><td>Glu</td><td>Thr</td><td>own</td><td>Pro</td><td>How much</td><td>Glu</td><td>Glu</td><td>Trp</td>

<td>Gln</td><td>His</td><td>How much</td><td>Glu</td><td>Gln</td><td>Leu</td><td>lys</td><td>Gly</td><td>Cheese</td><td>ala</td><td>Glu</td><td>Gly</td><td>Cheese</td><td>How much</td><td>Arg</td><td>Tyr</td>

<td>Pro</td><td>ala</td><td>Trp</td><td>own</td><td>Gly</td><td>How much</td><td>How much</td><td>Cheese</td><td>Gln</td><td>Glu</td><td>val</td><td>Leu</td><td>Asp</td><td>Tyr</td><td>Leu</td><td>Cheese</td>

<td>Cheese</td><td>Tyr</td><td>How much</td><td>own</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 50 <211> 1260 <212> DNA <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> Nucleotide sequence having preferred corn codons encoding FLP recombinase (FLPm) <221> CDS <222> (1) ... (1260) <400> 50

<td>atg Underworld 1</td><td>ccc Pro</td><td>cag Gln</td><td>ttc phe</td><td>gac Asp 5</td><td>atc How much</td><td>ctc Leu</td><td>TGC Cys</td><td>aag lys</td><td>acc Thr 10</td><td>ccc Pro</td><td>ccc Pro</td><td>aag lys</td><td>gtg val</td><td>ctc Leu 15</td><td>gtg val</td><td> 48</td>

<td>ctc</td><td>TGC</td><td>gcc</td><td>gcc</td><td>gag</td><td>ctc</td><td>acc</td><td>trays</td><td>ctc</td><td>TGC</td><td>TGG</td><td>atg</td><td>atc</td><td>acc</td><td>cac</td><td>aac</td><td> 144</td>

<td>GGC</td><td>acc</td><td>gcc</td><td>att</td><td>aag</td><td>agg</td><td>gcc</td><td>acc</td><td>ttc</td><td>atg</td><td>tCA</td><td>trays</td><td>aac</td><td>acc</td><td>atc</td><td>atc</td><td> 192</td>

<td>trays</td><td>aag</td><td>acc</td><td>cag</td><td>aag</td><td>gcc</td><td>acc</td><td>atc</td><td>ctc</td><td>gag</td><td>gcc</td><td>tcc</td><td>ctc</td><td>aag</td><td>aag</td><td>ctc</td><td> 288</td>

PZ / 1656 / AR atc ccc gcc tgg gag ttc acc atc atc ccc

Ile Pro Ala Trp Glu Phe Thr Ile Ile Pro

100 105 cag tcc gac atc acc gac atc gtg tca tcc

Gln Ser Asp Ile Thr Asp Ile Val Ser Cheese

115 120 tcc tcc gag gag gct gac aag ggc aac tcc

Cheese Cheese Glu Glu Ala Asp Lys Gly Asn Cheese

130 135 aag gcc ctc ctc tcc gag ggc gag tcc atc

Lys Ala Leu Leu Cheese Glu Gly Glu Cheese Ile

145 150 atc ctc aac tcc ttc gag tac acc tcc agg

Ile Leu Asn Ser Phe Glu Tyr Thr Ser Arg

165 170 ctc tac cag ttc ctc ttc ctc gcc acc ttc

Leu Tyr Gln Phe Leu Phe Leu Ala Thr Phe

180 185 lC3 yiC 3lC 33y 3.SC yyC GCC 33y tCC

Cheese Asp Ile Lys Asn Val Asp Pro Lys Cheese

195 200 aag tac ctc ggc gtg atc atc cag tgc ctc

Lys Tyr Leu Gly Val Ile Ile Gln Cys Leu

210 215 tcc gtg tcc agg cac atc tac ttc ttc tcc

Ser Val Ser Arg His Ile Tyr Phe Phe Ser

225 230 ccc ctc gtg tac ctc gac gag ttc ctc agg

Pro Leu Val Tyr Leu Asp Glu Phe Leu Arg

245 250 aag agg gtg aac agg acc ggc aac tcc tcc

Lys Arg Val Asn Arg Thr Gly Asn Cheese Ser

260 265 cag ctc ctc aag gac aac ctc gtg agg tcc

Gln Leu Leu Lys Asp Asn Leu Val Arg Ser

275 280 aag aac gcc ccc tac tcc atc ttc gcc atc

Lys Asn Ala Pro Tyr Cheese Ile Phe Ala Ile

290 295 cac atc ggt agg cac ctc atg acc tcc ttc

His Ile Gly Arg His Leu Met Thr Ser Phe

305 310 acc gag ctc acc aac gtg gtg ggc aac tgg

Thr Glu Leu Thr Asn Val Val Gly Asn Trp

325 330 gcc gtg gcc agg acc acc tac acc cac cag

EP 1 907 553 B1 trays ggc cag aag cac 336

Tyr Tyr Gly Gln Lys His 110 ctc cag ctt cag ttc gag 384

Leu Gln Leu Gln Phe Glu 125 cac tcc aag aag atg ctg 432

His Ser Lys Lys Met Leu 140 tgg gag atc acc gag aag 480

Trp Glu Ile Thr Glu Lys

155 160 ttc act aag acc aag acc 528

Phe Thr Lys Thr Lys Thr 175 'atc aac tgc ggc agg ttc 576

Ile Asn Cys Gly Arg Phe 190

-.- 4-4. ~ ~ £ 2 0/1 llu aay i-lu yuy na.y solid

Phe Lys Leu Val Gln Asn 205 gtg acc gag acc aag acc 672

Val Thr Glu Thr Lys Thr 220 gct cgc ggc agg atc gac 720

Ala Arg Gly Arg Ile Asp

235 240 aac tca gag ccc gtg ctc 768

Asn Ser Glu Pro Val Leu 255 tcc aac aag cag gag tac 816

Cheese Asn Lys Gln Glu Tyr 270 tac aac aag gcc ctc aag 864

Tyr Asn Lys Ala Leu Lys 285 aag aac ggc ccc aag tcc 912

Lys Asn Gly Pro Lys Ser 300 ctc tca atg aag ggc ctc 960

Leu Cheese Met Lys Gly Leu

315 320 tcc gac aag agg gcc tcc 1008

Ser Asp Lys Arg Ala Ser 335 atc acc gcc atc ccc gac 1056

VP / 1656 / AR

EP 1 907 553 B1

Ala Val Ala Arg Thr Thr Tyr Thr His Gln Ile Thr Ala Ile Pro Asp 340 345 350 cac tac ttc gcc ctc GTg tca agg tac tac gcc tac gac ccc atc tcc His Tyr Phe Ala Leu Val Ser Arg Tyr Tyr Ala Tyr Asp Pro Ile Cheese

355 360 365

1104

<td>lys</td><td>Glu</td><td>Underworld</td><td>How much.</td><td>ala</td><td>Leu</td><td>lys</td><td>Asp</td><td>Glu</td><td>Thr</td><td>own</td><td>Pro</td><td>How much</td><td>Glu</td><td>Glu</td><td>Trp</td>

<td>cag</td><td>cac</td><td>atc</td><td>gag</td><td>cag</td><td>ctc</td><td>aag</td><td>GGC</td><td>tcc</td><td>gcc</td><td>gag</td><td>GGC</td><td>tcc</td><td>atc</td><td>agg</td><td>trays</td>

<td>ccc</td><td>gcc</td><td>TGG</td><td>aac</td><td>GGC</td><td>atc</td><td>atc</td><td>tcc</td><td>cag</td><td>gag</td><td>gtg</td><td>ctc</td><td>gac</td><td>trays</td><td>ctc</td><td>tcc</td>

1152

1200

1248 tcc tac atc aac Ser Tyr Ile Asn

420

1260 <210> 51 <211> 78 <212> DNA <213> Artificial sequence <220>

<223> Oligonucleotide primer <221> misc_cecha <222> 38, 39, 40, 41, 42, 43 <223> n = A, T, C or G <400> 51 gccagcatgc aagcttgaat tccgaagttc ctatactnnn nnnagaatag gaacttcgag 60 gctggatcc 210> 52 <211> 78 <212> DNA

<213> Artificial sequence <220>

<223> Oligonucleotide primer <221> misc_cecha <222> 36, 37, 38, 39, 40, 41 <223> n = A, T, C or G <400> 52 cgttccgcgg atccagatct cgaagttcct attctnnnnn nagtatagga acttcggaat 60 tcaagggg 210> 53 <211> 23 <212> DNA <213> Artificial sequence <220>

<223> Oligonucleotide primer <400> 53 gcacatacaa atggacgaac gga 23 <210> 54 <211> 22 <212> DNA <213> Artificial sequence <220>

<223> oligonucleotide primer

PZ / 1656 / AR EP 1 907 553 B1 <400> 54 cctcttcgct attacgccag ct 22 <210> 55 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT6 site linker sequence <400> 55 tttttgaa 8 <210> 56 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT7 site linker sequence <400> 56 ttattgaa 8 <210> 57 <211> 8 <212> DNA <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> FRT22s linker sequence WO 01/23545 <400> 57 tatctaga 8 <210> 58 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT72s site linker sequence WO 01/23545 <400> 58 tttctaca 8 <210> 59 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> linker sequence of FRT3s site WO 01/23545 <400> 59 tatttgaa 8 <210> 60 <211> 8

PZ / 1656 / AR EP 1 907 553 B1 <212> DNA <213> Artificial sequence <220>

<223> FRT2161s linker sequence WO 01/23545 <400> 60 tctctgga 8 <210> 61 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT2151s site linker sequence WO 01/23545 <400> 61 tctccaga 8 <210> 62 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> linker sequence of FRT2272s site WO 01/23545 <400> 62 tatctaca

PZ / 1656 / AR EP 1 907 553 B1 <210> 63 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT2262s linker sequence WO 01/23545 <400> 63 tatcttga 8 <210> 64 <211> 8 <212> DNA <213> Artificial sequence <220>

<223> FRT2373s linker sequence WO 01/23545 <400> 64 tgtctata 8 <210> 65 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f22 WO 01/23545

PZ / 1656 / AR EP 1 907 553 B1 <400> 65 agttcctata ctatctagag aataggaact 30 <210> 66 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f72 WO 01/23545 <400> 66 agttcctata ctttctacag aataggaact 30 <210> 67 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f3 WO 01/23545.

<400> 67 agttcctata ctatttgaag aataggaact 30 <210> 68 <211> 30 <212> DNA <213> Artificial sequence

PZ / 1656 / AR EP 1 907 553 B1 <220>

<223> Minimum FRT site f2161 WO 01/23545 <400> 68 agttcctata ctctctggag aataggaact 30 <210> 69 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f2151 WO 01/23545 <400> 69 agttcctata ctctccagag aataggaact 30 <210> 70 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f2272 WO 01/23545 <400> 70 agttcctata ctatctacag aataggaact 30 <210> 71 <211> 30

PZ / 1656 / AR EP 1 907 553 B1 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f2262 WO 01/23545 <400> 71 agttcctata ctatcttgag aataggaact 30 <210> 72 <211> 30 <212> DNA <213> Artificial sequence <220>

<223> Minimum FRT site f2373 WO 01/23545 <400> 72 agttcctata ctgtctatag aataggaact

PZ / 1656 / AR EP 1 907 553 B1

Contents32

53 members in 9 offices

This recordMember, by publication datePriority claim

Priority claims6

Priority claims
Document	Office	Kind	Date
70022505	United States of America	P	2005-07-18
06787308	European Patent Office (EPO)	A	2006-07-14
2006027380	United States of America	W	2006-07-14
EP20060787308	–	–	–
US20050700225P	–	–	–
WO2006US27380	–	–	–

Members53

Family members, oldest first
Document	Office	Kind
US2007015195A1	United States of America	A1	2007-01-18
US2007016985A1	United States of America	A1	2007-01-18
CA2615797A1	Canada	A1	2007-01-25
WO2007011733A2	World Intellectual Property Organization (WIPO)	A2	2007-01-25
US2007178593A1	United States of America	A1	2007-08-02
WO2007011733A3	World Intellectual Property Organization (WIPO)	A3	2007-09-07
US2007271629A1	United States of America	A1	2007-11-22
CA2652598A1	Canada	A1	2007-11-29
WO2007137114A2	World Intellectual Property Organization (WIPO)	A2	2007-11-29
US2008047031A1	United States of America	A1	2008-02-21
WO2007137114A3	World Intellectual Property Organization (WIPO)	A3	2008-03-20
MX2008000764A	Mexico	A	2008-03-24
EP1907553A2	European Patent Office (EPO)	A2	2008-04-09
MX2008014615A	Mexico	A	2009-01-15
EP2018435A2	European Patent Office (EPO)	A2	2009-01-28
US2009100550A1	United States of America	A1	2009-04-16
US2009165176A1	United States of America	A1	2009-06-25
CN101490267A	China	A	2009-07-22
CA2736061A1	Canada	A1	2009-12-23
WO2009154639A1	World Intellectual Property Organization (WIPO)	A1	2009-12-23
US7736897B2	United States of America	B2	2010-06-15
US2010173801A1	United States of America	A1	2010-07-08
US2010192263A1	United States of America	A1	2010-07-29
BRPI0612862A2	Brazil	A2	2010-11-30
US2011047655A1	United States of America	A1	2011-02-24
MX2010014365A	Mexico	A	2011-03-15
EP2308986A1	European Patent Office (EPO)	A1	2011-04-13
EP2310516A1	European Patent Office (EPO)	A1	2011-04-20
US2011119795A1	United States of America	A1	2011-05-19
CN102131932A	China	A	2011-07-20
WO2007137114A8	World Intellectual Property Organization (WIPO)	A8	2011-12-01
EP2018435B1	European Patent Office (EPO)	B1	2012-07-11
EP1907553B1	European Patent Office (EPO)	B1	2012-08-22
BRPI0712398A2	Brazil	A2	2012-10-16
ES2390132T3	Spain	T3	2012-11-06
US8318493B2	United States of America	B2	2012-11-27
PL2018435T3	Poland	T3	2012-11-30
PL1907553T3This record	Poland	T3	2013-02-28
US2013052739A1	United States of America	A1	2013-02-28
CN101490267B	China	B	2013-04-17
CN103215304A	China	A	2013-07-24
US8586361B2	United States of America	B2	2013-11-19
CA2615797C	Canada	C	2014-04-29
EP2308986B1	European Patent Office (EPO)	B1	2014-10-08
US8900869B2	United States of America	B2	2014-12-02
US2015044771A1	United States of America	A1	2015-02-12
US9234194B2	United States of America	B2	2016-01-12
US2016108412A1	United States of America	A1	2016-04-21
BRPI0822806A2	Brazil	A2	2016-09-20
US9777284B2	United States of America	B2	2017-10-03
US2017369893A1	United States of America	A1	2017-12-28
US2019194673A1	United States of America	A1	2019-06-27
US11225668B2	United States of America	B2	2022-01-18

Modified frt recombination sites and methods of use

Abstract

Term

PZ / 1656 / AR EP 1 907 553 B1

PZ / 1656 / AR EP 1 907 553 B1

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EXPERIMENTAL PART

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Priority claims6

Members53

Numbers

Titles2

Classification