method to produce a transgenic plant or plant tissue

专利摘要:
ENGINEERED LANDING WRAPS TO DIRECT GENES ON PLANTS. The present invention relates to a method for producing a transgenic plant including providing a nucleic acid molecule comprising at least two nucleic acid sequence regions that lack sequence homology to the plant cell's genomic DNA, and at at least two zinc finger nuclease recognition sites, where the at least two nucleic acid sequence regions lacking sequence homology with plant cell genomic DNA span the at least two finger nuclease recognition sites zinc. A plant cell or tissue having a nucleic acid molecule stably integrated into the plant cell's genome is transformed. A plant is regenerated from the plant cell. Transgenic plants are produced by the method. The seeds are produced by transgenic plants.
公开号:BR112012018235B1
申请号:R112012018235-8
申请日:2011-01-21
公开日:2020-11-03
发明作者:William Michael Ainley；Ryan C. Blue；Michael G. Murray；David Richard Corbin；Rebecca Ruth Miles；Steven R. Webb
申请人:Down Agrosciences Llc；
IPC主号:

专利说明:

PRIORITY CLAIM
This application claims the benefit of the filing date of US Provisional Patent Application, serial number 61 / 297,641, filed on January 22, 2010, for "ENERGY-HARMFUL LANDED WRAPS FOR DIRECTING GENES IN PLANTS". DESCRIPTION FIELD
The present invention generally relates to compositions and methods for the generation of transgenic organisms, for example, plants. In certain embodiments, the methods of the invention allow for the incorporation of landing wraps (ELPs) for targeting at a genomic site, thereby further facilitating the introduction of nucleic acid molecules of interest into a defined genomic site containing one or more ELPs. In some embodiments, ELPS may comprise nucleotide sequence regions that essentially comprise random sequences flanking zinc finger nuclease (ZFN) binding sites. BACKGROUND
Many plants are genetically transformed with genes from other species to introduce desirable characteristics, such as to improve agricultural value through, for example, improving the quality of nutritional value, increasing productivity, providing resistance to pests or diseases, increasing tolerance to drought and tension, increase horticultural qualities such as pigmentation and growth, and / or confer resistance to herbicides, allowing the production of industrially useful compounds and / or materials from the plant, and / or enabling the production of pharmaceutical products. The introduction of cloned genes into plant cells and the recovery of stable fertile transgenic plants can be used to make such modifications to a plant and have enabled the genetic engineering of plants (for example, for crop improvement). In these methods, foreign DNA is typically randomly introduced into the nuclear or plastidial DNA of the eukaryotic cell, followed by isolation of cells containing the foreign DNA integrated into the cell's DNA, to produce stable transformed plant cells.
The first generations of transgenic plants were typically generated by Agrobacterium-mediated transformation technology. The success with these techniques has stimulated the development of other methods to introduce a nucleic acid molecule of interest in the genome of a plant, DNA absorption in protoplasts mediated by PEG, bombardment of microprojectiles and transformation mediated by crystalline silicon.
In all of these plant transformation methods, however, the transgenes incorporated into the plant's genome are integrated in a random manner and in unpredictable copy numbers. Often, transgenes can be integrated in the form of repeats, either of the whole or parts of it. Such complex integration pattern can influence the level of expression of the transgenes (for example, by destruction of the transcribed RNAs by means of silencing mechanisms of post-transcriptional genes, or by induction of methylation of the introduced DNA), thus regulating downward the transcription of the transgene. In addition, the integration site itself can influence the level of expression of the transgene. The combination of these factors results in a wide variation in the level of expression of the transgenes or foreign DNA of interest between cells of different transgenic plants and plant strains. In addition, the integration of the foreign DNA of interest can have a disruptive effect on the region of the genome where integration occurs and can influence or disrupt the normal function of the target region, thus leading to frequently undesirable side effects.
The foregoing requires that whenever the effect of introducing a particular foreign DNA into a plant is investigated, a large number of strains of transgenic plants are generated and analyzed, in order to obtain significant results. Likewise, in the generation of specimens of transgenic culture, in which a DNA of particular interest is introduced into plants to provide the transgenic plant with a desired phenotype, a large population of independently created transgenic plant strains is created to allow selection those strains of plants with optimal expression of the transgenes, and with minimal or no side effects on the global phenotype of the transgenic plant. Particularly in this field, more targeted transgenic approaches are desired, for example, in view of the heavy regulatory requirements and high costs associated with the repeated archived trials necessary to eliminate unwanted transgenic events. In addition, it will be evident that the possibility of insertion of target DNA will also be beneficial in the transgene stacking process.
Several methods have been developed in an effort to control the insertion of transgene in plants. See, for example, Kumar and Fladung (2001) Trends Plant Sci. 6: 155-9. These methods are based on transgene integration based on homologous recombination. This strategy has been successfully applied to lower prokaryotes and eukaryotes. Paszkowski et al. (1988) EMBO J. 7: 4021-6. However, for plants, until recently, the predominant mechanism for transgene integration is based on illegitimate recombination that involves little homology between the recombinant DNA strands. A major challenge in this area is, therefore, the detection of the rare events of homologous recombination, which are masked by the much more efficient integration of foreign DNA, introduced through illegitimate recombination.
Custom-designed zinc finger nucleases (ZFNs) are proteins designed to provide a double-stranded break in the targeted site-specific DNA, with subsequent recombination of the cleaved ends. ZFNs combine the nonspecific cleavage domain of a restriction endonuclease, such as, for example, Fok, with zinc finger DNA binding proteins. See, for example, Huang et al. (1996) J. Protein Chem. 15: 481-9; Kim et al. (1997) Proc. Natl. Acad. Sci. USA 94: 3616-20; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-60; Kim et al. (1994) Proc Natl. Acad. Sci. USA 91: 883-7; Kim et al. (1997b) Proc. Natl. Acad. Sci. USA 94: 12875-9; Kim et al. (1997c) Gene 203: 43-9; Kim et al. (1998) Biol.
Chem. 379: 489-95; Nahon and Raveh (1998) Nucleic Acids Res. 26: 1233-9; Smith et al. (1999) Nucleic Acids Res. 27: 674-81. Individual zinc finger motifs can be designed to target and link to a wide variety of DNA sites. Canonical Cys2HiS2, as well as non-canonical Cys3His zinc finger proteins link DNA by inserting an α helix into the main groove of the double helix. DNA recognition by zinc fingers is modular: each finger basically contacts three consecutive base pairs on the target, and some key residues in the protein mediate recognition. It has been shown that Fok restriction endonuclease must dimerize through the nuclease domain in order to cleave DNA, inducing a double strand break. Likewise, ZFNs also require dimerization of the nuclease domain in order to cut DNA. Mani et al. (2005) Biochem. Biophys. Commun. 334: 1191-7; Smith et al. (2000) Nucleic Acids Res. 28: 3361-9. Dimerization of the ZFN is facilitated by two adjacent, oppositely oriented binding sites. Id. DESCRIPTION SUMMARY
Here are described methods of introducing a nucleic acid molecule into the genome of a host organism that exhibits a low rate of homologous recombination, for example, a species of plant or animal. In particular examples, "engineered landing wraps (ELPs) are used, where ELPs can comprise nucleotide sequence regions comprising nucleotide sequences substantially devoid of homology to the host organism's genome (for example, sequences generated randomly) flanking binding sites of DNA binding domains (e.g., zinc finger proteins (ZFPs), meganucleases, or leucine zippers). DNA binding domains that target ELP binding sites can, naturally include functional DNA cleavage domains or can be part of fusion proteins that further comprise a functional domain, for example, an endonuclease cleavage domain or cleavage half-domain (for example, a ZFN, a recombinase, a transposase, or a housing endonuclease, including a housing endonuclease with a modified DNA binding domain.) This class of DNA binding and proton divage eins is collectively referred to here as "target endonucleases". In particular examples, non-randomized nucleotide sequences from biological sources other than the host organism can also be used; no homology to the genome of the target organism is adequate. Thus, in some examples, nucleotide sequences can be designed to ensure substantially no homology for the regions of any sequenced target plant genome.
In some instances, ELPs may provide regions of homology and binding sites for high activity for targeting target endonucleases (eg, ZFNs) for targeting homology-directed genes. Consequently, ELPs can reduce or eliminate the need to use other nucleotide sequences, external or internal, in a nucleic acid insert. ELPs can be designed to have no spurious open reading frames, and to contain or not contain restriction enzyme sites, as preferred, for vector construction or analysis of plants transformed with a nucleic acid molecule of interest. In some examples, ELPs may be flanked by non-identical restriction sites that generate compatible ends by digestion with restriction enzymes, but the linked hybrid sites are not cleavable by any of the restriction enzymes. In these and other examples, this allows concatenation of ELPS into larger matrices, each with unique regions of homology and targeting endonuclease binding sites (for example, the ZFN binding sites).
In some examples, ELPs may be randomly incorporated into a target genome, or targeted to genomic sites that have been shown to accommodate transgenic insertions without any, or with an acceptable harmful impact on the resulting transgenic plants. Regions of homology at the genomic target site may be identical to regions of homology in the target donor nucleic acid molecule, thus facilitating homology-oriented recombination after double-stranded dividing by targeting endonucleases (eg, ZFNs).
Consequently, methods of projecting and generating ELPs are described, as well as methods of using ELPs to transform a plant with a nucleic acid molecule of interest. Also described are nucleic acid molecules useful in the invention, and genetically modified plants produced by methods according to the invention.
The previous and other characteristics will become more evident from the following detailed description of various modalities, which continue with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 includes an illustration of a representative vector comprising an ELP, pDAB100610.
Figure 2 includes an illustration of a representative vector comprising an ELP, pDAB100611.
Figure 3 includes an illustration of a representative vector comprising an ELP, pDAB100640
Figure 4 includes an illustration of a representative vector comprising an ELP, pDAB100641
Figure 5 includes an illustration of a representative vector comprising a donor fragment to be integrated into an ELP, pDAB105955.
Figure 6 includes an illustration of a representative vector comprising a donor fragment to be integrated into an ELP, pDAB105956.
Figure 7 includes an illustration of a representative vector comprising a donor fragment to be integrated into an ELP, pDAB105979.
Figure 8 includes an illustration of a representative vector comprising a donor fragment to be integrated into an ELP, pDAB105980.
Figure 9 is a schematic diagram of: a) ELP 1 in maize; b) donor construct for redirecting ELP1; c) ELP1 site redirected.
Figure 10 includes an illustration of a representative vector pDAB104132.
Figure 11 includes an illustration of a DNA fragment representing a Multiple Cloning Site, which is flanked by constructed Zinc Finger binding sites and buffer sequences.
Figure 12 includes an illustration of a representative vector pDAB104126.
Figure 13 includes an illustration of a representative vector pDAB104136.
Figure 14 includes an illustration of a representative vector pDAB104138.
Figure 15 includes an illustration of a representative vector pDAB104140.
Figure 16 includes an illustration of a representative vector pDAB104142.
Figure 17 includes an illustration of a representative vector pDAB104133.
Figure 18 includes an illustration of a representative vector pDAB104134.
Figure 19 includes an illustration of a representative vector PDAB104135.
Figure 20 includes an illustration of a representative vector pDAB104137.
Figure 21 includes an illustration of a representative vector pDAB104139.
Figure 22 includes an illustration of a representative vector pDAB104141.
Figure 23 includes an illustration of a representative vector PDAB104143. DETAILED DESCRIPTION I. Overview of different modalities
In some embodiments, methods are provided for introducing nucleic acid molecules comprising "elaborate landing envelope" (ELPS) into a host organism, such that the integration of other nucleic acid molecules of interest to the organism host is facilitated. In embodiments, ELPs may comprise regions of nucleic acid sequence that are not homologous to native sequences of the host organism (for example, essentially randomly generated nucleic acid sequences, which can then be selected for insertion based on certain desirable criteria as described herein. ), flanking targeting endonuclease recognition sites (for example, ZFN recognition sites). Sites for the integration of ELPs can be random, they can be determined by identifying, for example, a structural gene within the host genome that has an expression of desired level for a nucleic acid molecule of interest to be introduced into the PLA site. , or it can be determined by identifying sites within the host genome that do not confer a metabolic, functional, agronomic (if a plant) or other penalty to the transformed host organism when the PLA is introduced into the site. ELPs can be introduced in tandem into the genome in such a way that, for example, ELPs are present as concatamers in an organism produced according to the methods of the invention.
In some modalities, the integration of a nucleic acid molecule of interest is performed at an ELP site. In embodiments, a nucleic acid molecule of interest to be introduced into an ELP site is associated with one or more targeting endonucleases, delivered as polypeptides or expressed from introduced RNA or DNA. In addition, the introduced nucleic acid molecule may include an ELP other than that already incorporated into the host genome. Those skilled in the art will realize that a nucleic acid sequence introduced into the host genome at a site where a native nucleic acid sequence is expressed in the wild-type organism is expected to be expressed in a similar way as the native nucleic acid sequence . For example, if the native nucleic acid sequence is expressed in the wild type organism under the control of regulatory elements (for example, such that, for example, the nucleic acid sequence is expressed in a tissue-specific manner or specific developmental), the introduced nucleic acid sequence is expected to undergo the same or similar regulatory control. In this way, ELPs can facilitate the desired temporal and / or spatial expression of a nucleic acid molecule of interest introduced into the ELP site. II. Abbreviations
ELISA immunoabsorbent assay linked to the ELP enzyme ZFN engineered grounding wrap zinc finger nuclease III. Terms
Gene expression: The process by which the encoded information of a transcriptional nucleic acid unit (including, for example, genomic DNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. The expression of the gene can be influenced by external signals, for example, the exposure of a cell, tissue or organism to an agent that increases or decreases the expression of the gene. Expression of a gene can also be regulated anywhere in the DNA to RNA pathway for the protein. Regulation of gene expression occurs, for example, through controls that act on the transcription, translation, transport and processing of RNA, degradation of intermediate molecules, such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules, after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro protein activity assay, in situ, or in vivo.
Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes hydrogen bonding by Watson-Crick, Ho-ogsteen or inverted Hoogsteen, between complementary bases. Generally, nucleic acid molecules consist of nitrogenous bases which are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the binding of pyrimidine to the purine is referred to as "base pairing." More specifically, A is linked by hydrogenide to T or U, and G will be linked to C. "Complementary" refers to the pairing of bases that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.
"Specifically hybridizable" and "specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the target DNA or RNA. The oligonucleotide does not need to be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when the binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is sufficient degree of complementarity to prevent non-specific binding of the oligonucleotide to non-target sequences under conditions where the specific binding is desired, for example, under physiological conditions, in the case of in vivo tests or systems. Such a link is referred to as specific hybridization.
Hybridization conditions, resulting in specific degrees of stringency will vary, depending on the nature of the hybridization method chosen and the composition and length of the hybridizing nucleic acid sequences. Generally, the hybridization temperature and ionic strength (especially the concentration of Na + and / or Mg2 +) of the hybridization buffer will contribute to the hybridization stringency, although washing times also influence the stringency. Calculations related to the hybridization conditions necessary to achieve particular degrees of strictness are discussed in Sambrook et al. (Ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, ch. 9 and 11.
For the purposes of the present description, "stringent conditions" encompass conditions under which hybridization will occur if there is at least 25% incompatibility between the hybridization molecule and the target sequence. "Strict conditions" can be further defined at certain levels of strict. Thus, as used here, "moderate stringency" conditions are those in which molecules with more than 25% incompatibility will not hybridize. "Medium stringency" conditions are those in which molecules with more than 15% incompatibility will not hybridize, and "high stringency" conditions are those under which sequences with more than 10% incompatibility will not hybridize. "Very high stringency" conditions are those under which sequences with more than 6% incompatibility do not hybridize.
In particular embodiments, stringent conditions may include hybridization at 65 ° C, followed by sequential washes, at 65 ° C with 0.1 x SSC / 0.1% SDS for 40 minutes.
Isolated: an "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced separately from, or purified from other biological components in the organism's cell in which the component occurs naturally, that is, another Chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules, proteins and peptides.
Nucleic acid molecule: a polymeric form of nucleotides, which can include both sense and antisense RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers from the above. A nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of any type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." The term includes single and double stranded forms of DNA. A nucleic acid molecule can include one, or both, naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds.
Nucleic acid molecules can be modified chemically or biochemically or can contain unnatural or derivatized nucleotide bases, as will be readily noted by those skilled in the art. Such modifications include, for example, labeling, methylation, and replacement of one or more of the naturally occurring nucleotides with an analog. Other modifications include internucleotide modifications, such as uncharged bonds (for example, methyl phosphonates, phosphotriets, phosphoramidates, carbamates, etc.), charged bonds (for example, phosphorothioates and phosphorodithioates, etc.), pending portions (for example, peptides tides), intercalators (eg acridine, psoralen, etc.), chelating agents, alkylators and modified bonds (eg alpha anomeric nucleic acids, etc.) The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairy, circular, and locked conformations.
Operationally linked: A first nu nucleic acid sequence is operationally linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked with a coding sequence when the promoter affects the transcription or expression of the coding sequence. When produced recombinantly, sequences of operably linked nucleic acids are generally contiguous and, when necessary, to join two protein coding regions in the same reading frame. However, the elements do not need to be contiguous to be operationally linked.
Promoter: a region of DNA that is usually located upstream (towards the 5 'region of a gene) that is needed for transcription. Prosecutors allow for the proper activation or repression of the gene they control. A promoter contains specific sequences that are recognized by transcription factors. These factors bind to the promoter DNA sequences and result in recruitment of RNA polymerase, the enzyme that synthesizes RNA from the gene's coding region. In some embodiments, tissue-specific promoters are used. A tissue-specific promoter is a DNA sequence that directs a higher level of transcription of an associated gene in the tissue to which the promoter is specific in relation to other tissues in the body. Examples of tissue-specific promoters include tapetum-specific promoters; anther-specific promoters; specific pollen promoters; (see, for example, U.S. Patent No. 7,141,424, and International PCT publication No. WO 99 / 042,587); specific egg promoters; (See, for example, U.S. Patent Application No. 2001/047525 A1); specific fruit promoters (see, for example, U.S. Patent No. 4,943,674, and 5,753,475), and specific seed promoters (see, for example, U.S. Patent No. 5,420,034, and 5,608,152). In some embodiments, development-specific promoters are also used, for example, an active promoter at a later stage in development.
Transformed: a virus or vector "transforms" or "transduces" a cell when it transfers nucleic acid molecules to the cell. A cell is "transformed" by a nucleic acid molecule transduced into the cell, when the nucleic acid molecule becomes stably replicated by the cell, either by incorporating the nucleic acid molecule into the cell genome or by episomal replication. As used herein, the term "transformation" encompasses all the techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation (Fromm et al. (1986) Nature 319: 791-3), lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7), microinjection (Mueller et al. (1978) Cell 15: 579-85), Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. USA 80: 4803-7), direct DNA absorption, and microprojectile bombardment (Klein et a /. (1987) Nature 327: 70).
Transgene: An exogenous nucleic acid sequence. In one example, a transgene is a sequence of genes (for example, a herbicide resistance gene), a gene that encodes an industrially or pharmaceutically useful compound, or a gene that encodes a desirable agricultural trait. In yet another example, the transgene is an antisense nucleic acid sequence, wherein the expression of the antisense nucleic acid sequence inhibits the expression of a target nucleic acid sequence. A transgene can contain regulatory sequences operatively linked to the transgene (for example, a promoter). In some embodiments, a nucleic acid molecule of interest to be introduced by a segmented ELP recombination is a transgene. However, in other embodiments, a nucleic acid molecule of interest is an endogenous nucleic acid sequence, in which additional genomic copies of the endogenous nucleic acid sequence are desired, or a nucleic acid molecule that is in the antisense orientation with respect to to a target nucleic acid molecule in the host organism.
Vector: a nucleic acid molecule as introduced into a cell, thereby producing a transformed cell. A vector can include nucleic acid sequences that allow them to be replicated in the host cell, such as an origin of replication. Examples include, but are not limited to, a plasmid, cosmid, bacteriophage, or virus that carries exogenous DNA in a cell. A vector can also include one or more genes, antisense molecules, and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express the nucleic acid molecules and / or proteins encoded by the vector. A vector can optionally include materials to assist in getting the nucleic acid molecule into the cell (for example, a liposome, protein coding, etc.). IV. Engineered ground wraps for gene targeting in plants A. Overview
In some embodiments, ELPS are introduced into plants, for example, to facilitate the introduction of one or more additional nucleic acid molecule (s) of interest. The nucleic acid molecule of additional interest can comprise, for example, any nu nucleic acid sequence that encodes a protein to be expressed in the host organism. In additional embodiments, ELPS are used to facilitate the introduction of nucleic acid molecules of unknown function to discern their function, based on ectopic gene expression. Regions flanking the ELPs can be homologous to the genomic nucleic acid sequence of the host plant, such that the ELPs are integrated into the genome of the host plant in a site-specific manner. ELPs can be used to incorporate nucleic acid molecules of various lengths.
In some embodiments, a nucleic acid molecule targeting a transgenic ELP target may contain a second ELP to allow continued gene addition at the target site. One or more target endonucleosis (for example, ZFNs) can be used sequentially or simultaneously in this process. If desired, more than one target endonuclease binding site (e.g., ZFN binding site) can also be incorporated internally into regions of the ELPs that are homologous to the host plant's genomic nucleic acid sequence. In some embodiments, targeting endonuclease binding sites can be added so that they flank the ELPS. In the latter and in still other modalities, the expression of a targeting endonuclease (for example, ZFN) that cleaves at the binding sites added in the plant leads to the excision of ELPS.
In some embodiments, regions homologous to the host plant's genomic nucleic acid sequence can also be used in combination with other DNAs that flank to allow insertion of nucleic acid molecules adjacent to a nucleic acid molecule of interest introduced into an ELP target, such as, for example, a gene expression element or gene of interest. DNA cleavage can be used to improve homologous recombination at this site. In some embodiments, regions of homology in ELPs that are homologous to the genomic nucleic acid sequence can also be used for targeted insertion of nucleic acid molecules that is facilitated by DNA-cleaving enzymes, such as ZFNs and meganucleases or other targeting endonucleases .
In some modalities, ELPs can be incorporated into modified chromosomal regions, so that they can be generated by DNA amplification processes or in minichromosomes. In some embodiments, regions of homology and targeting endonucleases properly placed (for example, ZFNs) are used to facilitate the modification of sequences internal to the flanking regions homologous to the genomic nucleic acid sequence of the host plant. B. Project of ELPs
In some embodiments, ELPs can be designed to facilitate homologous recombination at plant chromosomal sites. ELPs can have a neutral impact on surrounding genes or DNA sequences. ELPs can represent unique, segmentable sequences within the plant genome. In certain embodiments, the size (for example, 1 kb) for each 5 'and 3' homology region with a desired chromosomal location is selected to achieve the minimum size considered desirable to facilitate homology-oriented recombination. In specific embodiments, the size of each 5 'and 3' homology region is about 50 bp; 100 bp; 200 bp; 400 bp; 600 bp; 700 bp; 750 bp; 800 bp; 1 kb; 1.2 kb; 1.4 kb; 1.6 kb; 1.8 kb; 2 kb; or 3 kb. The 5 'and 3' homology regions need not be the same size in particular modalities. ELP sequences can be produced, for example, using a computer program to generate random numbers. ELP sequences produced, for example, by the generation of random sequence can then be selected for use as ELPs based on desirable characteristics including, but not limited to, sequences substantially or completely lacking homology with native genomic sequences of the target organism. Selected ELPs can also be modified as further detailed below.
In some embodiments, the criteria for designing ELPs and / or modifying selected ELPs may include one or more of the following: removal of restriction sites routinely used for cloning; reduction in the number of potential methylation sites (for example, CG and CNG); and absence of any open reading structure greater than 300 bp. In addition, during the design of ELPs, other sites in nucleic acid sequences can be removed, including, for example: exon junctions: intron (5 'or 3'); poly A plus signs; RNA polymerase termination signals; and highly stable secondary chain structures. Sequences can also be analyzed and modified to reduce the frequency of TA or CG doublets. Sequence blocks that have more than about six consecutive residues of [G + C] or [A + T] can also be removed from a sequence during the ELP project.
In embodiments, ELPs may include, for example, nucleotide sequences of the formula XI-Y-X2, where Y is at least one targeting endonuclease binding site (for example, a ZFN binding site), where Xi is a nucleotide sequence selected without homology to the host organism's genome and positioned 5 'from Y, and where X2 is a nucleotide sequence selected without homology to the host organism's genome that is different from Xi.
Exemplary nucleotide sequences Xi and X2 can be selected independently from the following sequences: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10. Additional modifications, deletions, or additions to these sequences (for example, removal, or the addition of restriction sites) can be done in some ways to provide additional or different functionality. For example, in certain modalities: SEQ ID NO: 1 can be modified to generate SEQ ID NO: 11; SEQ ID NO: 3 can be modified to generate SEQ ID NO: 12; SEQ ID NO: 2 can be modified to generate SEQ ID NO: 13; SEQ ID NO: 4 can be modified to generate SEQ ID NO: 14; SEQ ID NO: 5 can be modified to generate SEQ ID NO: 17; and SEQ ID NO: 6 can be modified to generate SEQ ID NO: 18.
In particular embodiments, exemplary ELPs include, but are not limited to, the following sequences: SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 19.
Restriction enzyme sites (for example, Fsel restriction enzyme sites) can be introduced by flanking an ELP, to allow cloning of the ELP into an appropriate vector. Restriction enzyme sites can also be introduced by flanking an ELP that produces compatible ends by fragmenting restriction enzymes (for example, BglW and BamHl sites), to allow the ELPs to be linked together, for example, as a concatamer, into the genome of the host plant. Restriction enzyme sites can also be introduced to allow analysis in the host plant of nucleic acid sequences of interest subsequently targeted to ELPS by recombination. Two or more restriction enzyme sites can be introduced by flanking a single ELP. For example, Bg / ll and BamHI sites can be included internally to Fsel sites. Restriction enzyme sites can also be introduced to allow analysis in the host plant of nucleic acid sequences of interest targeting the ELPs for insertion by recombination. For example, Pme sites can be introduced to flank the insertion site. C. Distribution of nucleic acid molecules in plant cells that contain an ELP
To allow endonuclease-mediated integration of targeting a nucleic acid molecule of interest (for example, ELP (s) and / or exogenous nucleic acid molecules targeting a previously integrated ELP) to a plant genome through integration specific, distribution of targeting endonucleases or nucleic acid molecules encoded by targeting endonuclease is required, followed by the expression of a functional targeting endonuclease protein in the plant cell. A nucleic acid molecule of interest will also be present in the plant cell, at the same time that the targeting endonuclease is distributed or expressed in such a way that the targeting functional endonuclease protein can induce double strand breaks in the (s) target site (s) that are then repaired by homology-activated integration of the nucleic acid molecule of interest to the target locus. The person skilled in the art can predict that the functional targeting endonuclease protein can be achieved by several methods, including, but not limited to, transgenesis of a construct encoding targeting endonuclease, or the transient expression of a construct encoding targeting endonuclease. In both cases, the expression of endonuclease-targeting functional protein and distribution of donor DNA in the plant cell can be carried out simultaneously, in order to trigger targeted integration.
Thus, in particular embodiments, targeting endonucleases (for example, ZFNs) are expressed from nucleic acid molecules in transformed plants to introduce double-strand breaks in the genome of plants transformed, for example, into one or more ELPS previously introduced to the plant. Endonuclease targeting can be used in a way that targets one or more recognition sequences designed to be present in ELPS. Design and selection approaches for building an ELP of the invention can thus begin by determining one or more sequences) of specific nucleic acid (s) that are recognized by targeting endonucleate (s) to be used ( s) subsequently to introduce a nucleic acid molecule of interest into the ELP. The flexibility and specificity of the ZFN system provides a level of control previously unattainable by known recombinase-mediated gene excision strategies.
As an example, ZFNs can be easily constructed, for example, to recognize specific nucleic acid sequences (for example, ELPSs. Wu et al. (2007) Cell. Mol. Life Sci. 64: 2933-44. coding for zinc finger recognition residues allows for the selection of new fingers that have high affinity for arbitrarily chosen DNA sequences, in addition, zinc fingers are natural DNA binding molecules, and constructed zinc fingers have shown act on their targets projected onto living cells, so nucleases based on zinc fingers are targetable to specific, but arbitrary, recognition sites.
The requirement for dimerization of the cleavage domains of chimeric zinc finger nu-cleases confers a high level of sequence specificity. Since each set of three fingers links nine consecutive base pairs, two chimeric nucleases effectively require an 18 bp target if each zinc finger domain has perfect specificity. Any given sequence of that length is expected to be unique within a single genome (assuming approximately 109 bp). Bibikova et al. (2001) Mol. Cell. Biol. 21 (1): 289-97; Wu et al. (2007), supra. In addition, additional fingers provide improved specificity, Beerli et al. (1998) Proc. Natl. Acad. Know. USA 95: 14628-33; Kim and Pabo (1998) Proc. Natl. A- cad. Know. USA 95: 2812-7; Liu et al. (1997) Proc. Natl. Acad. Know. USA 94: 5525-30, so that the number of zinc fingers in each DNA binding domain can be increased to provide even more specificity. For example, specificity can be further increased by using a pair of 4-finger ZFNs that recognize a 24 bp sequence. Urnov et al. (2005) Nature 435: 646-51. In this way, ZFNs can be used in such a way that a recognition sequence in an ELP introduced into the genome of the host plant is unique within the genome.
A nucleic acid molecule of interest that is introduced through specific recombination in an ELP can be operably linked to one or more plant (s) promoters by triggering the expression of the gene in an amount sufficient to confer a desired trait or phenotype. Suitable promoters for this and other uses are well known in the art. Non-limiting examples describing such promoters include U.S. Patent No. 6,437,217 (corn promoter RS81); 5,641,876 (actin rice promoter); 6,426,446 (RS324 corn promoter); 6,429,362 (PR-1 corn promoter); 6,232,526 (A3 corn promoter); 6,177,611 (maize promoters); 5,322,938, 5,352,605, 5,359,142, and 5,530,196 (35S promoter); 6,433,252 (corn L3 oleosin promoter); 6,429,357 (actin 2 rice promoter, and actin 2 rice intron); 5,837,848 (root specific promoter); 6,294,714 (light inducible promoters); 6,140,078 (salt-inducible promoters); 6,252,138 (pathogen inducible promoters); 6,175,060 (promoters inducible for phosphorus deficiency); 6,388,170 (bi-directional promoters); 6,635,806 (gamma-coxyline promoter) and U.S. Patent Application Serial No. 09 / 757,089 (corn chloroplast aldolase promoter). Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84 (16): 5745-9); octopine synthase promoter (OCS) (which is transported on tumor-inducing plasmids from Agrobacterium tumefacíens); kaolinimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. (1987) Plant Mol. Biol 9: 315-24); the CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-2; 35S promoter of the Figwort mosaic virus (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84 (19): 6624- 8); the sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87: 4144-8); the R gene complex promoter (Chandler et al. (1989) Plant Cell 1 : 1175-83); the promoter of the a / b chlorophyll binding protein gene; CaMV35S (US Patent No. 5,322,938, 5,352,605, 5,359,142, and 5,530,196); FMV35S (US Patent No. 6,051. 753, and 5,378,619); a PC1SV promoter (US Patent No. 5,850,019), the SCP1 promoter (US Patent No. 6,677,503), and AGRtu.nos promoters (GenBank No. Adhesion V00087 ;. Depicker et al. (1982) J. Mol. Appl. Genet 1: 561-73 ;. Bevan et al. (1983) Nature 304: 184-7), and the like.
Additional genetic elements that can optionally be operatively linked to a nucleic acid molecule of interest include sequences that encode transit peptides. For example, the incorporation of a suitable transit chloroplast peptide, such as A. thaliana EPSPS CTP (Klee et al. (1987) Mol. Gen. Genet 210: 437-42), and Petunia hybrida EPSPS CTP (della- Cioppa et al. (1986) Proc. Natl. Acad. Sci. USA 83: 6873-7) has been shown to point sequences of heterologous EPSPS proteins to chloroplasts in transgenic plants. Dicamba monoxygenase (DMO) can also be oriented towards chloroplasts, as described in PCT International publication No. WO 2008/105890.
Additional genetic elements that can optionally be operatively linked to a nucleic acid molecule of interest also include 5 'RTU's located between a promoter sequence and a coding sequence that function as a leader translation sequence. The leading translation sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The leading translation sequence can affect the processing of the primary mRNA transcript, the stability of the mRNA and / or the efficiency of the translation. Examples of leading translation sequences include maize and petunia heat shock protein leaders (U.S. Patent No. 5,362,865), plant virus capital protein leaders, rubisco plant leaders, and others. See, for example, Turner and Foster (1995) Molecular Biotech. 3 (3): 225-36. Non-limiting examples of 5 'RTUs include GmHsp (U.S. Patent No. 5,659,122); PhDnaK (U.S. Patent No. 5,362,865); AtAntl; TEV (Carrington and Freed (1990) J. Virol 64: 1590-7.); and AGRtunos (GenBank N ° Adhesion V00087; and Bevan et al. (1983) Nature 304: 184-7).
Additional genetic elements that can optionally be operatively linked to a nucleic acid molecule of interest also include 3 'untranslated sequences, 3' transcription termination regions, or polyadenylation regions. These are genetic elements located downstream of a polynucleotide molecule, and include polynucleotides that provide a polyadenylation signal, and / or other regulatory signals capable of affecting transcription, mRNA processing, or gene expression. The polyadenylation signal functions in plants to cause the addition of polyadenylated nucleotides to the 3 'end of the mRNA precursor. The polyadenylation sequence can be derived from the natural gene, from a variety of plant genes, or from T-DNA genes. A non-limiting example of a 3 'termination region of transcription is the 3' region of nopaline synthase (NOS 3 '; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80: 4803-7). An example of the use of different 3 'untranslated regions is provided in Ingelbrecht et al., (1989) Plant Cell 1: 671-80. Non-limiting examples of polyadenylation signals include one from a Pisum sativumRbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984) EMBO J. 3: 1671-9) and AGRtu.nos (GenBank No. Adhesion E01312). D. Transformation of host cells with nucleic acid molecules
Any of the techniques known in the art, for introducing nucleic acid molecules into plants, can be used to produce a transformed plant according to the invention, for example, to introduce one or more ELPS into the genome of the host plant, and / or to further introduce a nucleic acid molecule of interest. Suitable methods for transforming plants include any method by which DNA can be introduced into a cell, such as: by electroporation, as illustrated in U.S. Patent No. 5,384,253; by bombardment of microprojectiles, as illustrated in U.S. Patent Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865; by Agrobacterium-mediated transformation, as illustrated in U.S. Patent Nos. 5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301, and by transformation of protoplasts, as set forth in U.S. Patent No. 5,508,184. Through the application of techniques like these, the cells of virtually any plant species can be stably transformed, and these cells can be grown in transgenic plants using techniques known to those skilled in the art. For example, techniques that may be particularly useful in the context of cotton processing are described in U.S. Patent Nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344; techniques for transforming Brassica plants in particular are described, for example, in U.S. Patent No. 5,750,871; techniques for transforming soy are described, for example, in U.S. Patent No. 6,384,301; and techniques for processing corn are described, for example, in U.S. Patent No. 7,060,876, U.S. Patent No. 5,591,616, and PCT International Publication WO 95/06722.
After making the distribution of exogenous DNA to recipient cells, the transformed cells can be identified, in general, for later culture and plant regeneration. In order to improve the ability to identify transformants, one may wish to employ a selectable or traceable marker gene with the transformation vector used to generate the transformant. In this case, the population of potentially transformed cells can be assayed by exposing the cells to an agent, or selective agents, or the cells can be screened for the desired marker gene characteristic.
Cells that survive exposure to the selective agent, or cells that have been positively scored in a screening assay, can be grown in media that support plant regeneration. In some embodiments, any suitable plant tissue culture media (for example, MS and N6 media) can be modified by including other substances, such as growth regulators. The tissue can be kept in a basic medium with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or by following repeated rounds of manual selection, until the tissue morphology is suitable for regeneration (eg example, at least 2 weeks), then transferred to media conducive to the formation of sprouts. The cultures are transferred periodically until sufficient budding has occurred. Once the shoots are formed, they are transferred to means conducive to the formation of roots. Once sufficient roots are formed, the plants can be transferred to the soil for further growth and maturity.
To confirm the presence of a nucleic acid molecule of interest in regenerating plants, a variety of assays can be performed. Such assays include, for example: molecular biological assays, such as by Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISA and / or Western blots) or by enzymatic function; assays of plant parts, such as leaf or root assays, and analysis of the phenotype of the whole regenerated plant.
Targeted integration events can be selected, for example, by PCR amplification using, for example, specific initiating oligonucleotides for nucleic acid molecules of interest. PCR genotyping is understood to include, but is not limited to, polymerase chain reaction (PCR) amplification of genomic DNA derived from tissues isolated from host plant calluses expected to contain a nucleic acid molecule of interest integrated into the genome, followed by standard cloning and sequence analysis of PCR amplification products. PCR genotyping methods have been well described (for example, Rios, G. et al. (2002) Plant J. 32: 243-53) and can be applied to genomic DNA derived from any plant species or tissue type, including cell cultures. Combinations of oligonucleotide primers that bind to both the target sequence and the introduced sequence can be used sequentially or multiplexed in PCR amplification reactions. Oligonucleotide initiators designed to anneal to the target site, introduced nucleic acid sequences, and / or combinations of the two are feasible. Thus, PCR genotyping strategies may include (but are not limited to) amplification of specific sequences in the plant genome, amplification of multiple specific sequences in the plant genome, amplification of non-specific sequences in the plant genome, or combinations thereof . The person skilled in the art may notice additional combinations of primers and amplification reactions to interrogate the genome. For example, a set of forward and reverse oligonucleotide primers can be perceived to anneal the target-specific nucleic acid sequence (s) outside the limits of the introduced nucleic acid sequence.
Forward and reverse oligonucleotide primers can be designed to anneal specifically to an intruded nucleic acid molecule of interest, for example, to a sequence corresponding to a coding region within the nucleic acid molecule of interest, or other parts of the nucleic acid molecule of interest. These primers can be used in conjunction with the primers described above. Oligonucleotide Primers can be synthesized according to a desired sequence, and are commercially available (for example, from Integrated DNA Technologies, Inc., Coralville, IA). Amplification can be followed by cloning and sequencing, or by analyzing the direct sequence of the amplification products. The person skilled in the art can envisage alternative methods for the analysis of the amplification products generated during PCR genotyping. In one embodiment, oligonucleotide primers specific for the target gene are employed in PCR amplifications. E. Cultivation and use of transgenic plants
A transgenic plant that includes one or more ELPs and / or nucleic acid molecule of interest, inserted by recombination at the ELP site according to the present invention, can have one or more desirable characteristics. Such characteristics may include, for example: resistance to insects, pests and other disease-causing agents; herbicide tolerance; greater stability, yield, or expiration date; environmental tolerances; pharmaceutical production, industrial product production, and nutritional improvements. Desirable characteristics can be conferred by one or more nucleic acid molecules inserted by specific recombination in an ELP site that are expressed in the plant exhibiting desirable characteristics. Thus, in some embodiments, the desired characteristic may be due to the presence of a transgene in the plant, which is introduced into the plant's genome at the site of an ELP. In an additional embodiment, the desirable characteristic can be obtained through conventional reproduction, whose characteristic can be conferred by one or more nucleic acid molecules inserted (s) by specific recombination in an ELP site.
A transgenic plant according to the invention can be any plant capable of being transformed with a nucleic acid molecule of the invention. Consequently, the plant can be a dicot or monocot. Non-limiting examples of dicotyledonous plants usable in the present methods include alfalfa, beans, broccoli, cabbage, carrots, cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant, lettuce, melon, peas, pepper, peanuts, potatoes, pumpkin, radish, canola, spinach, soy, pumpkin, beet, sunflower, tobacco, tomato and watermelon. Non-limiting examples of monocot plants usable in the present methods include maize, onion, rice, sorghum, wheat, rye, corn, sugar cane, oats, triticale, grass, and grass.
The transgenic plants according to the invention can be used or grown in any form, in which the presence of ELP (s) and / or nucleic acid molecules of interest is desirable. Consequently, they can be designed to, inter alia, have one or more desired characteristics, when being transformed with nucleic acid molecules according to the invention, and cut and cultured by any method known to those skilled in the art. EXAMPLES
The following examples are included to illustrate embodiments of the invention. It will be appreciated by those skilled in the art that the techniques described in the Examples represent the techniques discovered by the inventors to work well in the practice of the invention. However, those skilled in the art, in light of the present description, will appreciate that many changes can be made in the specific modalities that are described and still obtain a similar or similar result, without departing from the scope of the invention. More specifically, it will be evident that certain agents that are both chemically and physiologically related can be replaced by the agents described herein, while the same or similar results would be achieved. All such similar substitutes and apparent modifications to those skilled in the art are considered to be within the scope of the invention as defined by the appended claims. Example 1: vector construction
Two different ELPS were synthesized which consisted of a 5 kbp 5 'homology region ("engined landing wrap region 1" (SEQ ID NO: 11) in p DAB100610 / pDAB 100640 and "homology region 3 'engineered synthetic "(SEQ ID NO: 12) in pDAB100611 / pDAB100641) and a 1 kbp homology 3 region (" engineered landing wrap region 2 "(SEQ ID NO: 13) in pDAB100610 and" homology region " 4 'targeted synthetic (SEQ ID NO: 14) in pDAB 100611 / pDAB 100641). These regions of homology were separated by two different EXZACT Nuclease zinc finger (eZFN) binding sites.
The two ELPS used for pDAB100610 and pDAB100611, designated ELP1 (SEQ ID NO: 15) and ELP2 (SEQ ID NO: 16), respectively, were transferred using the Gateway ® LR clonase reaction (Invitrogen, Carlsbad, CA) in pDAB100704, a superbinary target vector that is derived from pSB11 (WQ94 / 00977 and WO95 / 06722). pDAB100704 contains the ZmUbil promoter (promoter, 5 'untranslated region (UTR) and intron derived from ubiquitin's Zea mays 1 promoter (Chistensen et al., (1992) Plant Molecular Biology, 18 (4); 675-89)), the selectable marker gene AAD-1 (a synthetic, plant-optimized version of a Sphingobium herbicidovorans dioxygenase aryloxyalkanate gene (ATCC ® 700,291) that encodes an enzyme with ketoglutarate-dependent alpha dioxigenase activity that confers resistance to aryloxyphenoxypropionate herbicides (WO 2005/107437, WO 2008 / 141154A2, and USA 2.009 / 0.093.366, each of which is incorporated herein by reference), and the 3 'ZmLip RTU (3' untranslated region (RTU), comprising the transcription terminator and polyadenylation site of the LIP gene Zea mays; GenBank adhesion L35913).
The two ELPS used for pDAB 100640 and pDAB 100641, designated ELP1 (SEQ ID NO: 15) and ELP2 (SEQ ID NO: 16), respectively, were transferred using the Gateway ® LR clonase reaction (Invitrogen, Carlsbad, CA) in pDAB101849, a binary vector, pDAB101849 contains the OsActl promoter (promoter, and intron derived from the rice actin promoter (US Patent No. 5,641,876)), the selectable marker gene pat (phosphinothricin acetyl transferase gene; Wohlleben et al. , (1988) Gene 70: 25-37), and 3 'ZmLip RTU (3' untranslated region (RTU), comprising the transcription terminator and polyadenylation site of the LIP gene Zea mays; GenBank adhesion L35913).
The resulting clones, pDAB100610 (figure 1), pDAB100611 (figure 2), pDAB100640 (figure 3), and pDAB100641 (figure 4) were checked for the sequence. The vectors pDAB100610 and pDAB100611 were transferred to an Agrobacterium tumefaciens strain LBA4404 embracing the binary plasmid pSB1, and the T chain region was integrated into pSB1 via homologous recombination. The vectors pDAB100640 and pDAB100641 were transferred to an Agrobacterium tumefaciens LBA4404 strain. The structure of each plasmid was confirmed by fragmentation with restriction enzymes.
Vectors containing nucleic acid molecules of interest (donor DNA) were assembled by inserting a YFP expression cassette and either the PAT or AAD-1 expression cassette in the middle of truncated versions of 5 'and 3' homology regions ( noted as versions 2 (v2)) for ELP1 and ELP2. The PAT expression cassette contains the OsActl promoter (rice actin promoter; McElroy et al., (1990) Plant Cell 2: 163-171) that was used to express a pat gene (phosphinothricin acetyl transferase gene; Wohlleben et al ., (1988) Gene 70: 25-37) which is flanked by an untranslated 3 'ZmLip region. The AAD-1 expression cassette contains the ZmUbi 1 promoter to trigger the aad-1 gene and the 3 'ZmPer 5 UTR (U.S. Patent No. 6,384,207). The YFP expression cassette consists of the ZmUbil promoter that is used to trigger the expression of the PhiYFP gene (yellow fluorescent protein (US Patent Application 2007/0298412)) and is flanked by the 3 'ZmPer5 RTU (US Patent No. 6,384,207) .
The expression cassettes PAT and YFP were cloned between the truncated versions of "engineered landing wrap region 1" and "landing pad engineering region 2" in ELP1 (pDAB105955, figure 5). Alternatively, the expression cassettes PAT and YFP were cloned between the truncated versions of "built homology region 3" and "engineered homology region 4" in ELP2 (pDAB105956, figure 6). Likewise, the expression cassettes AAD-1 and YFP were cloned between the truncated versions of "engineered landing wrap region 1" and truncated versions of "engineered landing wrap region 2" in ELP1 (pDAB105979, figure 7) . Alternatively, the expression cassettes AAD-1 and YFP were cloned between the truncated versions of "engineering homology region 3" and truncated versions of "engineering homology region 4" in ELP2 (pDAB105980, figure 8). The donor DNA constructs were cloned into a binary vector by biolistic transformation, SILÍCIO CRISTALINO or mediated by Agrobacterium. In addition, DNA constructs can be cloned into a high number of copies of the plasmid (for example, pBR322 derivatives having a Co-IE1 origin of replication) to generate DNA from the plasmid used for direct transformation of DNA delivery. Example 2: Crystalline Silicon-mediated DNA distribution
Cultures of embryogenic corn Hi-ll cells were produced as described in U.S. Patent No. 7,179,902, and were used as a source of live plant cells in which specific integration was exemplified.
Fragments containing the selectable plant marker cassette AAD-1 and ELP1 and ELP2, respectively, of pDAB100610 and pDAB100611 and fragments containing the plant selectable marker cassette PAT and ELP1 and ELP2, respectively, of pDAB100640 and pDAB100641 were used for generate transgenic events. Transgenic events have been isolated and characterized. These events were then targeted using homologous homologous recombination, in which a nucleic acid molecule of interest can be integrated into the engineering landing pad. 12 ml of cell concentrate volume (PCV) from a previously cryopreserved cell line plus 28 ml of conditioned medium was subcultured into 80 ml of GN6 liquid medium (N6 medium (Chu et al., (1975) Sci Sin. 18: 659-668), 2.0 mg / L of 2,4-D, 30 g of sucrose / L, pH 5.8) in a 500 mL Erlenmeyer flask, and placed on a shaker at 125 rpm at 28 ° C This step was repeated twice using the same cell line, such that a total of 36 mL of PCV was distributed into three vials. After 24 hours, the GN6 liquid media was removed and replaced with 72 ml of GN6 S / M osmotic medium (N6 medium, 2.0 mg / 1 2,4-D, 30 g / L sucrose, 45, 5 g / L of sorbitol, 45.5 g / L of mannitol, 100 mg / L of myo-inositol, pH 6.0). The flask was incubated in the dark for 30-35 minutes at 28 ° C with moderate agitation (125 rpm). During the incubation period, a 50 mg / L suspension of capillary silicon carbide crystals (Advanced Composite Materials, LLC, Greer, SC) was prepared by adding 8.1 mL of GN6 S / M liquid medium to 405 mg crystalline silicon carbide silicon carbide.
After incubation in GN6 S / M osmotic medium, the contents of each flask were collected in a 250 ml centrifuge bottle. After all the cells in the flask had settled to the bottom, the volume of excess content of approximately 14 mL of GN6 S / M liquid was removed and collected in a 1 L sterile flask for future use. The crystalline silicon pre-moistened suspension was mixed at a maximum vortex speed for 60 seconds, and then added to the centrifuge bottle. 170 pg of purified DNA fragment pDAB100610 plasmid or pDAB100611 was added to each vial. Once the DNA was added, the bottle was immediately placed in a modified Red Devil 5400 commercial ink mixer (Red Devil Equipment Co., Plymouth, MN), and stirred for 10 seconds. After stirring, the cocktail of cells, media, crystalline silicon and DNA was added to the contents of a 1 L flask, along with 125 mL of new GN6 liquid medium to reduce the osmotic. The cells were left to recover in an agitator set at 125 rpm for 2 hours. Six milliliters of dispersed suspension were filtered on Whatman # 4 filter paper (5.5 cm) using a glass cell collecting unit connected to a vacuum line of its own such that 60 filters were obtained per flask. The filters were placed on 60 x 20 mm plates of solid GN6 medium (the same as GN6 liquid medium, except with 2.5 g / L of Gelrite gelling agent) and cultured at 28 ° C under dark conditions for 1 week. Example 3: Identification and isolation of putative transgenic events
One week after DNA release, filter papers were transferred to 60 X 20 mm plates of GN6 (1H) selection medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / L sucrose, 100 mg / L of myo-inositol, 2.5 g / L of Gelrite, pH 5.8) containing the appropriate selective agent. These selection plates were incubated at 28 ° C for one week, in the dark. After 1 week of selection in the dark, the tissue was incorporated into new media by scraping 14 of the cells from each plate into a tube containing 3.0 ml of GN6 agarose medium maintained at 37-38 ° C (medium N6, 2, 0 mg / L 2,4-D, 30 g / L of sucrose, 100 mg / L of myo-inositol, 7 g / L of SeaPlaque ® agarose, pH 5.8, autoclaved for 10 minutes at 121 ° C).
The agarose / tissue mixture was broken with a spatula and subsequently 3 ml of agarose / tissue mixture was evenly poured onto the surface of a 100 x 15 mm petri dish containing GN6 medium (1H). This process was repeated for both halves of each plate. Once all the tissue was incorporated, the plates were individually sealed with NESCOFILM ® or PARAFILM ® M, and cultured at 28 ° C, under dark conditions for up to 10 weeks. Putatively transformed isolates that grew under these selection conditions were removed from the embedded plates and transferred to new selection medium in 60 x 20 mm plates. If sustained growth was evident after about 2 weeks, an event was considered to be resistant to the applied herbicide (selective agent) and an aliquot of cells was subsequently harvested for genotype analysis. Example 4: molecular characterization of events
Extraction of genomic DNA. Genomic DNA (gDNA) was extracted from isolated corn cells and used as templates for PCR genotyping experiments. gDNA was extracted from approximately 100-300 pL of concentrated cell volume (PCV) of Hi-ll callus which was isolated according to the manufacturer's protocols detailed in DNeasy ® 96 Plant Kit (QIAGEN Inc., Valencia, CA). Genomic DNA was eluted in 100 pL of elution buffer provided with the kit giving final concentrations of 20-200 ng / pL, and subsequently analyzed using PCR-based genotyping methods. Molecular analysis of number of copies.
The determination of the number of transgenic copies by the hydrolysis probe assay, analogous to the TaqMan ® assay, was performed by real-time PCR using the L1GHTCYCLER480 ® system (Roche Applied Science, Indianapolis, IN). The assays were designed for the AAD-1 and PAT genes and an internal reference gene using LightCycler ® Design Probe Software 2.0. For amplification, a LightCycler ® 480 Probes Master mixture (Roche Applied Science, Indianapolis, IN) was prepared in 1x final concentration in a 10 pL volume multiplex reaction containing 0.4 pM from each primer and 0.2 pM from each probe. A two-stage amplification reaction was performed with an extension at 58 ° C for 38 seconds with the acquisition of fluorescence. All samples were performed in triplicate and the mean Cycle (Ct) limit values were used for the analysis of each sample. Real-time PCR data analysis was performed using LightCycler ® software version 1.5 using the relative quant module and was based on the ΔΔCt method. For this, a genomic DNA sample from a single known copy calibrator and 2 copy verification was included in each series (identical to those used by the INVADER ® assays above). From these data, the apparent copy number of AAD-1 or PAT was then estimated for each sample.
Initiator design for PCR genotyping.
Oligonucleotide primers were synthesized (for example, by Integrated DNA Technologies, Inc. (Coralville, Iowa)) under standard desalination conditions and diluted with water to a concentration of 100 pM. The oligonucleotide primer was designed for annealing to the end regions of the DNA insert. Primers were tested using dilutions of plasmid DNA in the presence of DNA isolated from non-transgenic organisms. The insertion of pDAB100610, pDAB100611, pDAB100640 and pDAB100641 was amplified by PCR from the genomic DNA of the putative events using the primers. The resulting fragment was cloned into a plasmid vector and sequenced to confirm that the engineered landing envelope was fully integrated into the plant's genome during transformation. Southern Blot analysis.
Southern analysis was performed to confirm the number of copies of the transgene. For this analysis, the genomic DNA was fragmented with suitable restriction enzymes and probed.
The corn tissue identified as containing a putative ELP by site-specific PCR was advanced for Southern blot analysis. For Southern analysis, tissue samples were collected in 2ml eppendorf tubes and lyophilized for 2 days. Maceration of the tissue was performed with a Kleco tissue sprayer and tungsten granules (Kleco, Visalia, CA). After tissue maceration, genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the protocol suggested by the manufacturer.
Genomic DNA was quantified using the Quant-IT Pico Green DNA assay kit (Molecular Probes, Invitrogen, Carlsbad, CA). The quantified DNA was adjusted to 4 pg for Southern blot analysis and fragmented with appropriate restriction enzymes overnight at 37 ° C. The fragmented DNA was purified using Quick-Precip (Edge Biosystem, Gaithersburg, MD) according to protocol suggested by the manufacturer. The precipitated DNA was resuspended in 10X dye and subjected to electrophoresis for 17 hours on a 0.8% SeaKem LE agarose gel (Lonza, Rockland, ME) at 40 volts. The DNA was transferred to loaded nylon membranes (Millipore, Bedford, MA) overnight and cross-linked to the membrane using the Strata UV 1800 ligand (Stratagene, La Jolla, CA). The membranes were prehybridized with 20 ml of PerfectHyb Plus (Sigma, St. Louis, MO) and probed with an appropriate probe labeled by radio overnight. The membranes were washed and placed on phosphor image screens for 24 hours and then analyzed using a STORM ™ 860 scanner (Molecular Dynamics). Example 5: Selection of transgenic events with insertion DNA
Events were selected by hydrolysis probe and PCR assay, as described above, for an intact plant transcriptional unit (PTU) containing AAD-1 or PAT gene cassettes and intact ELPs. The copy number was further confirmed by Southern analysis using standard methods with an AAD-1 and PAT gene and an ELP probe. Selected calluses from transgenic events that harbor a single copy, intact inserts of pDAB100610 and pDAB100611 were maintained for subsequent testing for AAD-1 gene expression. Screening using an AAD-1 qRT-PCR and ELISA method (below) identified events that express AAD-1.
QRT-PCR analysis of events that express AAD-1.
Quantitative real-time PCR (qRT-PCR) was used to quantify the mRNA expression of the AAD-1 gene expression. The assay was developed to quantify the relative expression of AAD-1 mRNA from samples of Hi-ll transgenic calluses by normalizing these levels against mRNA expression from an internal reference gene. Normalization of AAD-1 mRNA against mRNA from an internal reference gene allows comparison of AAD-1 expression between different samples, and can be used to identify events that appear to be highly expressive.
Total RNA was prepared from new callus tissue using Qiagen RNeasy ® 96 Kit (Qiagen, Valencia, CA). The RNA was treated with DNase without RNAse according to the kit instructions to remove any contaminants from genomic DNA. Synthesis of the first chain was created according to the instructions of the manufacturer of Supercript® III Reverse Transcriptase Enzyme (Invitrogen, Carlsbad, CA) and prepared using random hexamers. The synthesized cDNA strands were diluted in water in 1:10 and 1:50 proportions (this provides sufficient model for PCR amplification of multiple targets). Each aliquot was stored at -20 ° C indefinitely. QRT-PCR reaction mixtures were created for the amplification of AAD-1 cDNA as follows: 7.5 pl of 2X LC480 Probes Master Buffer (Roche Diagnostic, Indianapolis, IN), 0.3 pL of specific gene primer a from 10 pM of stock, 0.3 pL of gene specific direct primer from 10 pM of stock, 0.15 pL of LightCycler ® 480 Probes Master UPL probe, Roche Diagnostic, Indianapolis, IN), 1.5 pl of 10% (w / v) of polyvinyl pyrrolidone-40 (PVP-40), and 3.9 pL of water. The UPL probe (Roche Diagnostics, Indianapolis, USA) was one of blocked nucleic acid and, therefore, has a higher Tm than otherwise calculated. All components were put back in the freezer before dealing with patterns and unknowns. A 384-well microplate was demarcated and labeled, 13.5 pL of master mix was added per well. A sealing sheet was gently attached to the microplate. The plate was centrifuged for 1 minute at 3000 rpm in a Qiagen microplate centrifuge. 1.5 pl thawed diluted synthesized cDNA strands were added. In addition, 1.5 ul of DNA plasmid copy number standards were added to separate wells in a series of dilutions from the lowest to the highest concentration, these standards were compared with the AAD-1 cDNA (synthesized from total mRNA ) to quantify the number of copies. AAD-1 DNA from standard copy number series were made by cloning the target amplified fragment into a pCR2.1 plasmid (Invitrogen, Carlsbad, CA) and making a series of dilutions to quantify the number of copies. An aluminum seal was firmly attached to the plate and spun as described above. A PCR program was performed and the DNA was amplified in real-time PC4 LC480 instrumentation (Roche, Indianapolis, IN) or equivalent. Example 6: Determination of AAD-1 proteins in maize tissues using ELISA
A method to quantitatively determine the AAD-1 protein in corn calluses using an enzyme linked immunosorbent assay (ELISA) technique has been developed. The method described here can be used to detect AAD-1 protein and analyze tissue samples from transgenic corn callus plants.
The AAD-1 protein was extracted from corn samples with specific callus buffers based on phosphate buffered saline containing 0.05% Tween 20 (PBST) and possibly containing bovine serum albumin, protease inhibitors or Ascorbic acid. The extract was centrifuged, the aqueous supernatant collected, diluted and assayed using a specific ELISA for AAD-1. A simultaneous ELISA sandwich format was applied in this assay. An aliquot of the diluted sample and a monoclonal biotinylated anti-AAD-1 antibody (MAb 473F185) was incubated in the wells of a microtiter plate coated with an immobilized anti-AAD-1 monoclonal antibody (MAb 473H274). These corn-bound antibodies expressed the AAD-1 protein in the wells to form a "sandwich" with AAD-1 proteins bound between soluble antibodies and the immobilized one. Unbound samples and conjugate were then removed from the plate by washing with PBST. An excess amount of streptavidin enzyme conjugate (alkaline phosphatase) was added to the incubation wells. The presence of AAD-1 was detected by incubating the enzyme conjugate with an enzyme substrate; generating a colorful product. Since AAD-1 was bound in the antibody sandwich, the level of color development is related to the concentration of AAD-1 in the sample (ie, lower concentrations of protein result in lower color development). The absorbance at 405 nm was measured using a plate reader.
High AAD-1 expression events were identified from the selected transgenic events and were maintained for subsequent targeting with donor DNA. Example 7: Biolistics-mediated DNA distribution in plant cells that contain an ELP
Regeneration of transgenic events with target DNA Events of pDAB100160 and pDAB100611 confirmed to be low copy and containing intact PTU were regenerated to produce immature embryo donor material for targeting. Healthy growth tissue was first transferred to 28 + 100 haloxyfop (MS medium (Murashige and Skoog (1962) Physiol Plant 15: 473-497), 0.025 mg / L 2,4-D, 5 mg / L BAP, 0 , 0362 mg / L of haloxyfop, 30 g / L of sucrose, 2.5 g / L of gelrite, pH 5.7) and incubated in low light (14 pE / m2 ~ * "after 6 h of photoperiod) for 7 consecutive days with high light (89 pE / m2 • sec 16 h of photoperiod) for another 7 days, greening structures were transferred to 36 + 100 haloxyfop (same as 28 + 100 haloxyfop minus BAP and 2,4-D) and incubated in high light (40 pE / m2 • sec 16 h photoperiod) until sprout structures developed sufficient roots to transplant to the greenhouse The plants were grown in the greenhouse until maturity using the 95% Metro-Mix 360 ® mixture and 5% of the clay / clay soil and pollinated depending on the health of the plant. Plants that grow vigorously were self-fertilized or sibbed (plants from the same event) and the less vigorous plants were crossed with Hi-II, A188 or B104 to maintain the embryogenic capacity of donor material. Example 8: Biolistics-mediated DNA distribution in plant cells that contain an ELP
Direction of events produced from pDAB 100610 epDABI 00611.
Targeting the donor sequence was completed using two different transformation protocols. For intact transgenic ELPs that contained AAD-1 as a selectable marker (events pDAB100610 and pDAB100611), targeting was performed by delivery of mediated DNA by biolytic in immature pre-callous embryos. Embryos 1.2-2.0 mm in size were harvested 10-13 days after pollination and plated in N6E medium (N6 medium, 2.0 mg / 1 of 2.4-D, 2.8 g / L of L- proline, 100 mg / L of casein hydrolyzate, 100 mg / L of myo-inositol, 4.25 mg / L of silver nitrate, 30 g / L of sucrose, pH 5.8) and incubated at 28 0 C for 2-3 days in the dark. Swollen embryos were transferred to N6OSM (the same as N6E with the addition of 36.4 g / 1 of sorbitol, 36.4 g / L of mannitol and L-proline reduced to 0.7 g / L) which filled a circle 2 , 5 cm in diameter on Whatman # 4 filter paper. A 1000 pm screen was used to contain the embryos for pickling and embryos were incubated in the dark at 28 ° C for 4 hours before pickling. To coat the biological particles with DNA, 3 mg of 0.60 micron diameter gold particles were washed once with 100% ethanol, twice with sterile distilled water, and resuspended in 50 pl of water in a microcentrifuge tube. simulated. A total of 5 pg of separate plasmid DNA vectors encoding zinc finger nuclease and donor DNA fragment; pDAB105955 or pDAB105956), 20 pl of spermidine (0.1 M) and 50 pl of calcium chloride (2.5 M) were added separately to the gold suspension and mixed in a vortex. The mixture was incubated at room temperature for 10 min, pelleted at 10,000 rpm in a bench microcentrifuge for 10 seconds, resuspended in 60 pl of 100% cold ethanol, and 8-9 pl were distributed in each macro-carrier.
The bombing took place using the PDS-1000 / HE ™ bio-system (Bio-Rad Laboratories, Hercules, CA). Plates containing the embryos were placed in the middle of the shelf under conditions of 650 psi and 27 inches of Hg ++ in a vacuum, and were bombarded once following the operating manual. Twenty-four hours post-bombardment the embryos were transferred directly (without filter paper) to N6E (the same as above) for recovery and incubated for 13 days at 28 ° C in the dark. The embryos were transferred to N6S selection medium (N6 medium, 2.0 mg / 1 of 2,4-D, 100 mg / L of myo-inositol, 0.85 mg / L of silver nitrate, 2 mg / L of bialaphos, 30 g / 1 of sucrose, 2.5 g / L of gelrite and pH 5.8) for every 2 weeks during 3 transfers in selection medium and incubated at 28 ° C in dark conditions for up to 10 weeks. Putatively transformed isolates that grew under these selection conditions were removed from the embedded plates and transferred to fresh selection medium in 60 x 20 mm plates. If sustained growth was evident after approximately 2 weeks, an event was considered to be resistant to the applied herbicide (selective agent) and an aliquot of cells was subsequently harvested for genotype analysis (analysis described below).
Direction of events produced from pDAB100640 and pDAB100641.
For intact transgenic PLAs that contained PAT as a selectable marker (pDAB 100640 and pDAB100641), it was carried out via bio-mediated DNA delivery in corn embryogenic calluses. From six to eight days after subculture, the embryogenic corn tissue ((approximately 0.4 mL of PCV cells) was finely dispersed in a 2.5 cm diameter circle on top of Whatman # 4 filter paper placed on a plate 100 x 15 mm petri dish containing GN6 S / M medium (N6 medium, 2.0 mg / L 2,4-D, 30 g / 1 sucrose, 45.5 g / L sorbitol, 45.5 g / L of mannitol, 100 mg / L of myo-inositol, pH 5.8) solidified with 2.5 g / L of gelrite. The cells were incubated in the dark for 4 hours. DNA was prepared for pickling as previously described, pDAB105979 and pDAB105980 were used for targeting.
The bombing took place using the PDS-1000 / HE ™ biolistics system (Bio-Rad Laboratories, Hercules, CA). Plates containing the cells were placed on the middle shelf under conditions of 1,100 psi and 27 inches of Hg ++ vacuum, and were bombarded once following the operating manual. Twenty-four hours post-bombardment, the filter paper containing the plant cells was transferred to solid GN6 medium (N6 medium, 2.0 mg / L 2,4-D, 100 mg / L myo-inositol, 30 g / L of sucrose, pH 5.8) solidified with 2.5 g / L of Gelrite and incubated for 24 hours at 28 ° C under dark conditions. After the 24-hour recovery period, the filter paper containing the plant cells was transferred to GN6 + 100 haloxyfop (N6 medium, 2.0 mg / L 2,4-D, 100 mg / L myo-inositol , 30 g / 1 of sucrose, 0.0362 mg / L of haloxyfop, 2.5 g / L of Gelrite, pH 5.8), spreading the cells in a thin layer on the filter paper. Transfers continued every 2 weeks for 3 transfers in a selection medium. The tissue was incubated for up to 12 weeks until isolated putative transgenics resulting from the integration of donor DNA began to appear. Putatively transformed isolates that grew under these selection conditions were removed from the embedded plates and transferred to fresh selection medium in 60 x 20 mm plates. If sustained growth was evident after approximately 2 weeks, an event was considered to be resistant to the applied herbicide (selective agent) and an aliquot of cells was subsequently harvested for genotype analysis (analysis described below). Example 9: Screening for targeted integration events through PCR genotyping
Targeting of donor molecules (pDAB105979, pDAB105980, pDAB105955, pDAB105956) in ELPs in transgenic maize is analyzed using a combination of: 1) Site-specific PCR; 2) TaqMan from specific donor, and, 3) Southern blots from specific site to assess insertion accuracy, completeness and number of copies. A redirected positive event is expected to have a) a result of outside-outside PCR and overlapping inside-outside PCR and 2) a Southern image diagnosis of the presence of a donor. DNA extraction
Redirected transgenic corn tissue (callus tissue or plant leaves) is lyophilized for at least 2 days in 96-well collection plates (Qiagen, Germantown, MD). DNA is extracted from lyophilized tissue using a BioSprínt 96 workstation (Qiagen, Germantown, MD), following the manufacturer's instructions and resuspended in 200 pl of water. A Kleco 2-96A fabric sprayer (Garcia Manufacturing, Visalia, CA) is used for tissue rupture.
DNA Quantification: Resulting genomic DNA is quantified using a QUANT-IT ® Pico GReen DNA assay kit (Molecular Probes, Invitrogen Carlsbad, CA.). Five pre-quantified DNA standards ranging from 20 ng / pL to 1.25 ng / pL (diluted in series) are used to generate the standard curve. Unknown samples are first diluted in 1:10 or 1:20 dilutions to be within the linear range of the assay. 5 pl of diluted samples and standards are mixed with 100 pL of diluted Pico Green substrate (1: 200) and incubated for ten minutes in the dark. The fluorescence is then recorded using a Synergy2 plate reader (BioTek). The concentration of genomic DNA is estimated from the standard curve calculated after background fluorescence corrections. The DNA is subsequently normalized to concentrations of 2 ng / pL using a Biorobot-3000 automated liquid manipulator (Qiagen, Germantown, MD). Normalized DNA is used for PCR and copy number analysis. PCR analysis
Three types of site-specific PCR (outside-outside, 5 'outside-in, 3' outside-in) are performed to assess whether donor plasmids are targeted to ELPs. PCR reactions are performed to investigate the presence of an intact copy of the donor DNA (outside-outside PCR). Additional PCR reactions focus on the 5 'boundary between target and donors and the 3' boundary between donor and target (inside-outside PCR). A schematic with the positions of the primers used for the analysis is shown in figure 9. The expected PCR products for each donor and target ELP are described in Table 1. Table 1. The sequences of primers used for the analysis are shown in Table 2. Table 1 : Amplified fragment sizes expected for specific site PCR for integration analysis of segmented donors.

PCR amplification:
The polymerase chain reaction (PCR) is performed to assess and confirm ELPS redirection. PCR reactions are prepared from a 25 pl volume with DNA Fusion polymerase (New England Biolabs, Ipswich, MA) or AccuPrime (Invitrogen, Carlsbad, CA). For each Phusion PCR reaction, it contains 1X Phusion GC buffer, 0.2 mM dNTPs, 0.2 pM sense and reverse oligos, 2.5 units of Phusion DNA polymerase and 20 ng of genomic DNA. Ten to twenty ng of plasmid DNA constructed to mimic a redirected event is performed as a positive control. A non-model control (water as a model) is also performed. The PCR conditions are as follows: 35 cycles of 98 ° C, 1 min, 98 ° C 10 sec, 65 ° C, 20 sec, 72 ° C for 2.5 minutes followed by 72 ° C for 10 min.
Twenty-five microliters of PCR reaction performed with Ac-cuPrime (Invitrogen, Carlsbad, CA) contained 1X buffer II, 0.2 pM sense and reverse oligos, 2.5 units of AccuPrime Taq polymerase, and 20 ng of genomic DNA . Positive and negative controls are performed as described above for Phusion. Conditions for AccuPrime are as follows: 35 cycles of 98 ° C, 1 min, 98 ° C 10 sec, 65 ° C, 20 sec, 68 ° C for 2.5 minutes followed by 68 ° C for 10 min.
The amplified fragments are excised and purified with gel according to the manufacturer's instructions. Purified fragments are subsequently cloned into a plasmid vector and transformed into competent Escherichia coli cells.
The individual colonies are selected and confirmed to contain the fragment amplified by PCR. Double chain sequencing reactions from plasmid clones are performed to confirm that the PCR amplified genomic sequence contains the integrated donor. Events identified to contain the donor fragment represent a target in which homology-triggered repair of a ZFN-mediated double strand break and specific integration of a DNA donor into a specific target gene has occurred.
Hydrolysis probe / TaqMan hydrolysis assay TaqMan analysis is performed to define the number of donor copies in putative redirected events. Determination of the number of copies per hydrolysis probe assay, analogous to the TaqMan ® assay, is performed by real-time PCR using the LIGHTCYCLER ® 480 system (Roche Applied Science, Indianapolis, IN). The assays are designed for PAT and AAD1 and the internal reference gene GLP1 and Invertase using LIGHTCYCLER ® Probe Design Software 2.0. For amplification, LIGHTCYCLER ® 480 Probes Master mixture (Roche Applied Science, Indianapolis, IN) is prepared in 1X final concentration in a 10 pL volume multiplex reaction containing 0.4 35 cycles of 98 0 C, 1 min, 98 ° C 10 sec, 65 ° C, 20 sec, 72 ° C for 2.5 minutes followed by 72 ° C for 10 min. M of each primer and 0.2 35 cycles of 98 ° C, 1 min, 98 ° C 10 sec, 65 ° C, 20 sec, 72 ° C for 2.5 minutes followed by 72 ° C for 10 min. M for each probe (Table 3). The two-step amplification reaction is performed with an extension at 58 0 C for 38 seconds for PAT / GLP1 and 60 0 C for 40 seconds for AAD-1 and Invertase with fluorescence acquisition. All samples are run in triplicate and the average Cycle (Ct) limit values are used for the analysis of each sample.
Real-time PCR data analysis is performed using the LIGHTCYCLER © version 1.5 software using the relative quant module and is based on the ΔΔCt method. For this, a sample of gDNA from a single copy of calibrator and verification of known copy 2 was included in each series. Table 3: Initiator and probe information for pat hydrolysis probe test and internal reference (HMG)
The Southern analysis
Southern analysis is performed to confirm the targeting of donor DNA at the ELP site. For this analysis, the genomic DNA was digested with appropriate restriction enzymes and probed with a radiolabeled fragment from a specific site (not present in the donor).
Corn calluses (pDAB100640 and pDAB100641 target experiments) or T0 corn plants (pDAB100610 and pDAB100611 target experiments) are used. The tissue samples are collected in 2 ml microcentrifuge tubes and lyophilized for 2 days. Maceration of the tissue is performed with a Kleco tissue sprayer and tungsten granules as above. After tissue maceration, the genomic DNA is isolated using DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the protocol suggested by the manufacturer.
Genomic DNA is quantified by Quant-lt Pico Green DNA assay kit (Molecular Probes, Invitrogen, Carlsbad, CA). Four micrograms of DNA for each sample are fragmented with appropriate restriction enzymes overnight at 37 ° C. The fragmented DNA is purified using Quick-Precip (Edge Biosystem, Gaithersburg, MD) according to the protocol suggested by the manufacturer. After electrophoresis on a 0.8% SeaKem LE agarose gel (Lonza, Rockland, ME), the DNA is transferred to loaded nylon membranes (Millipore, Bedford, MA) overnight and cross-linked to the membrane using the ligand Strata UV 1800 (Stratagene, La Jolla, CA). Blots are pre-hybridized with 20 ml of PerfectHyb Plus (Sigma, St. Louis, MO) and probed with an appropriate radio-labeled probe overnight. The blots are washed and placed on phosphor imaging screens for 24 h and then analyzed using a Storm 860 digitizer (Molecular Dynamics). Example 10: Construction of an intermediate plant transformation vector that will accept ELPS, selectable markers, and are enabled for in-plant excision of independent construct elements
Segmented zinc finger nuclease binding sites (eZFN) and ELP elements can be incorporated, individually or together, into plant transformation vectors such that the transformed plant cells, tissues or resulting plants will have the following properties: 1) the ability to be re-transformed with additional transgenes in a precise and focused manner inside the PLA at the original transgenic insertion site; and 2) the ability to modify transgenic sites by removing transgenes, particularly selectable plant marker genes, from a predictable and efficient way. A series of vectors was built to transform a range of plant species. This was achieved through the use of an "intermediate plant transformation vector" (described below), a vector to affect incorporation of eZFNs flanking all selectable and ELPS markers into base vectors known as primary transformation vectors.
An intermediate plant transformation vector was engined that comprised a main chain of Agrobacterium binary transformation vector with a single multiple cloning site (MCS) positioned between the right and left edges of T-DNA. This intermediary vector is pDAB104132 (figure 10). The MCS used in pDAB104132 contains unique restriction enzyme sites for Agel, Notl, Fsel, Swal, Xbal, Asei, Pad, Xhol, Sail, and Nsil. In this example, these sites are used to incorporate the main functional elements into the final primary transformation vectors. The single Notl site can be used to introduce a Notl fragment by loading a Gateway ™ target vector cassette, which can then be subsequently used to stack multiple transgenes into the transformation vector using Invitrogen's GATEWAY ™ system. The single Fsel site can be used to introduce ELPS that have Fsel sites at their termini. As described earlier in these examples, when present in a transgenic plant, ELPs can be used as target sites to insert additional genes using eZFNs. Other sites in the MCS can be used to insert other genes of interest, including selectable marker genes. Using the unique restriction sites described above, intermediate plant transformation vectors such as pDAB104132 can be used to unite any and all combinations of functional elements into a single plant transformation vector, including GATEWAY ™ target vector cassettes, ELPS, selectable ZFN marker modules, and any other transgenes that may or may not be enabled for eZFN excision. Example 11: Construction of selectable plant marker cassettes enabled for excision using zinc finger nuclease modules.
In this example, eZFN binding sites are used to allow in-plant elimination of any transgene, including selectable marker genes, from a transformed plant. See US Provisional Patent Application No. _61 / 297,628, incorporated herein by reference. This ability is achieved by flanking transgenes with one or more eZFN binding sites and then incorporating these excisable gene modules into transformation vectors which are then used to generate transgenic plant cells. Construction of such modules and vectors can be performed by one skilled in the art using standard molecular biology techniques.
An intermediate plasmid carrying a ZFN binding site module is a useful first step in the construction of excisable gene cassettes. An eZFN binding site module is a segment of DNA that contains at least two eZFN binding sites that flank one or more restriction enzyme sites into which transgenes can be cloned. An example is the ZFN 2: 4-MCS-2: 4 module represented by SEQ ID: 20 and figure 11. This module contains a multiple cloning site (MCS) consisting of restriction sites Asei, Hindlll, Pad, Sbfl, Sphl and Sail that are flanked by pairs of binding sites for eZFN2 and eZFN4. Also within this ZFN module are two identical copies of a sequence consisting of a 100 bp random sequence that border the eZFN binding sites. In addition, pairs of Spel and Xhol restriction enzyme sites flank the entire functional module, allowing it to be cloned into a plant transformation vector. pDAB104126, a plasmid carrying the ZFN 2: 4-MCS-2: 4 module is shown in figure 12.
Four different selectable plant marker genes were individually inserted into the ZFN 2: 4-MCS-2: 4 module in pDAB104126 using standard molecular cloning techniques. A selectable marker gene was a functional PAT gene designed to confer tolerance to glufosinate in dicot plants. This PAT gene was composed of a PAT coding sequence operably linked to a CsVMV promoter (promoter and 5 'untranslated region derived from the Cassava Vein Mosaic virus; Verdaguer et al. (1996) Plant Molecular Biology 31 (6): 1129-1139 ) and the AtuORFI 3 'RTU. The PAT gene cassette was inserted as a Hindlll-Sphl fragment into the Hindlll-Sphl sites of the ZFN module to give plasmid pDAB104136 (Figure 13).
A second selectable marker gene was a PAT gene, designed to confer tolerance to glufosinate in monocot plants. This PAT gene comprised a PAT coding sequence operably linked to an OsActl promoter and a 3 'ZmLip RTU. This monocotyledon PAT gene was inserted as a Pacl-Sphl fragment into the Pacl-Sphl sites of the ZFN module to render plasmid pDAB104138 (figure 14).
A third selectable marker gene composed of an AAD-1 gene designed to confer tolerance to haloxifop or 2,4-D in monocotyledonous plants. This AAD-1 gene, composed of an AAD-1 coding sequence operatively linked to the ZmUbil promoter and a 3 'ZmLip RTU. This AAD-1 monocotyledon gene was inserted as a Hindlll-Sbfl fragment into the Hindlll-Sbfl sites of the ZFN module to give plasmid pDAB104140 (Figure 15).
A fourth selectable marker gene comprised a PAT gene designed to confer tolerance to glufosinate in canola plants. This PAT gene comprised a PAT coding sequence operably linked to a CsVMV promoter and an AtuORFI 3 'RTU and placed within a disrupted isopentenyl transferase (IPT) gene. The PAT gene was inserted as an Asel-Sall fragment into the Ase-Xhol sites of the ZFN module to render plasmid pDAB104142 (figure 16). Thus, pDAB104136, pDAB104138, pDAB104140 and pDAB104142 are intermediate plasmids that carry different excision-enabled ZFN selectable marker genes that can be incorporated into plant transformation vectors. When finally incorporated into a plant genome by transformation, the ZFN selectable marker modules allow for the subsequent removal of the selectable marker gene. This can be achieved through the production of eZFN2 or eZFN4 proteins in plant cells, either by transient expression or stable expression of genes that encode them. For example, eZFN2, or alternatively eZFN4, can be expressed in plant cells previously transformed by transient eZFN expression from any eZFN2 or eZFN4 genes. EZFN enzymes will recognize and bind specific eZFN binding sites and cause double strand breaks to occur in the genome at these positions. Double-strand breaks on both sides of the selectable marker gene within the module will result in excision of the selectable marker gene from the genome's DNA. Thereafter, the double chain break will be repaired, either by joining non-homologous end or homologous single chain repair between the repeated 100 bp sequences that were included in the ZFN module. This process will result in permanent exclusion of the selectable marker gene for its original genomic location. Example 12: Assembly of primary plant transformation vectors.
One skilled in the art can assemble the elements described above step by step in combination to produce primary transformation vectors for use in different crop plants. ELP1 was inserted into the Fsel site of pDAB104132 to give pDAB104133 (figure 17). Next, a Gateway ™ target vector cassette was inserted into the Notl site of pDAB104133 to yield pDAB104134 (figure 18) and pDAB104135 (figure 19) that differ only in their orientation from the Gateway cassette relative to the ELP sequence. Then, the four selectable ZFN marker modules described above were cloned into pDAB104135 to produce four new Plant Transformation Vectors (PTV). This assembly can be performed by anyone skilled in the art using standard DNA cloning methods as described below.
The dicotyledonized PAT ZFN module was excised from pDAB104136 using Spel and cloned into the Xbal site of pDAB104135 to give PTV pDAB104137 (figure 20). pDAB104137 is a binary plant transformation vector that carries ELP1 and a selectable excisable dicotyledon PAT ZFN marker, and is also a GATEWAY ™ target vector that allows the Gateway ™ cloning of additional transgenes.
The monocotyledon PAT ZFN module was excised from pDAB104138 using Spel and cloned at the Xbal site of pDAB104135 to give PTV pDAB104139 (figure 21). pDAB104139 is a binary plant transformation vector carrying ELP1 and a selectable excisable monocotyledon PAT ZFN marker, and is also a GATEWAY ™ destination vector.
The monocotylated ZFN AAD1 module was excised from pDAB104140 using Spel and cloned into the Xbal site of pDAB104135 to give PTV pDAB104141 (figure 22). pDAB104141 is a binary plant transformation vector carrying ELP1 and a selectable ZFN excisable monocotylated AAD1 marker, and is also a Gateway ™ target vector
The canola PAT ZFN module was excised from pDAB104142 using Xhol and cloned into the Xhol site of pDAB104135 to give PTV pDAB104143 (figure 23). pDAB 104143 is a binary plant transformation vector carrying ELP1 and a selectable excisable canola PAT marker ZFN, and is also a GATEWAY ™ target vector.
These constructs can be used to transform crop plants, using plant transformation methods. The resulting transgenic events can subsequently be the target of additional new transgenes. Wherein, the new transgene is segmented through homologous recombination with ELPS. In addition, the selectable marker expression cassette can be removed or excised. The resulting ELP that is produced from excision of the selectable marker cassette can subsequently be redirected with a new transgene, through homologous recombination, using the techniques described above.

权利要求:
Claims (9)
[0001]
1. Method for producing a transgenic plant or plant tissue, characterized by the fact that it comprises: providing a nucleic acid molecule comprising at least two nucleic acid sequence regions that lack sequence homology with the genomic DNA of the host plant, and at least one targeting endonuclease recognition site, where the two nucleic acid sequence regions that lack substantial sequence homology to the host plant cell's genomic DNA flank at least one site recognition of the targeting endonuclease, in which the two nucleic acid sequence regions that lack sequence homology to the host plant cell's genomic DNA do not comprise an open reading frame; where the two nucleic acid sequence regions that lack sequence homology to the host plant cell's genomic DNA are selected from one or more of the group consisting of the sequence ID NOs: 1 to 14, 17 or 18, or the two nucleic acid sequence regions that lack sequence homology with the genomic DNA of the host plant cell together with the targeting endonuclease sites comprise Sequence IDs 15, 16 and 19; transforming the plant cell or plant tissue comprising the plant cell with the nucleic acid molecule, wherein the nucleic acid molecule is stably integrated into the genome of the plant cell; and generating plant tissue or regenerating a plant from the plant cell transformed with the nucleic acid molecule.
[0002]
Method according to claim 1, characterized by the fact that the nucleic acid molecule additionally comprises non-identical restriction sites, preferably non-identical restriction sites comprise compatible single-stranded ends that allow landing wraps to weather ( Multiple ELPs).
[0003]
Method according to claim 1 or 2, characterized in that the at least two nucleic acid sequence regions that lack sequence homology with the plant cell's genomic DNA are from about 50 bp to about 3 kb.
[0004]
Method according to any of claims 1 to 3, characterized in that the targeting endonuclease site is a zinc finger nuclease site.
[0005]
Method according to any one of claims 1 to 4, characterized in that the nucleic acid molecule is either stably integrated randomly into the plant cell genome, or it is stably integrated into one or more known target sites in the plant cell genome, or is stably integrated into an amplified region of a chromosome or a minichromosome.
[0006]
Method according to any one of claims 1 to 5, characterized in that the nucleic acid molecule is flanked at each end by one or more additional targeting endonuclease recognition sites.
[0007]
Method according to claim 6, characterized in that it further comprises introducing into the plant or plant tissue one or more targeting endonucleases that recognize the one or more additional targeting endonuclease recognition sites, in which the Nucleic acid is excised from the genome of the plant or plant tissue.
[0008]
8. Method for producing a transgenic plant, characterized by the fact that it comprises: providing a cell or tissue isolated from the tissue of the transgenic plant or plant as defined in claim 3; providing at least one targeting endonuclease or a first nucleic acid molecule comprising a nucleic acid sequence encoding at least one targeting endonuclease, wherein the at least one targeting endonuclease (s) recognizes the, at least one targeting endonuclease recognition site introduced into the genome of the transgenic plant; providing a second nucleic acid molecule comprising a nucleic acid sequence of interest, and two additional nucleic acid sequences that flank the nucleic acid sequence of interest, wherein each of the two additional nucleic acid sequences is homologous with one of the two nucleic acid sequence regions that lack sequence homology to genomic DNA; introducing (i) the at least one targeting endonuclease or the first nucleic acid molecule, and (ii) the second nucleic acid molecule in the plant or tissue cell, where the nucleic acid sequence of interest is stably integrated into the plant cell genome; and regenerating a plant from the plant cell or tissue transformed with the first and second nucleic acid molecules.
[0009]
Method according to claim 7 or 8, characterized in that the targeting endonuclease is a zinc finger nuclease.

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法律状态:
2018-03-06| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-09-03| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-03| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/01/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US29764110P| true| 2010-01-22|2010-01-22|

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US61/297,641|2010-01-22|

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PCT/US2011/022145|WO2011091317A2|2010-01-22|2011-01-21|Engineered landing pads for gene targeting in plants|

---