Claims for Patent: 8,829,275

Summary for Patent: 8,829,275

Title:	Methods and compositions for obtaining marker-free transgenic plants
Abstract:	The invention provides methods and compositions for identifying transgenic seed that contain a transgene of interest, but lack a marker gene. Use of an identification sequence that results in a detectable phenotype increases the efficiency of screening for seed and plants in which transgene sequences not linked to a gene of interest have segregated from the sequence encoding a gene of interest.
Inventor(s):	Ye; Xudong (Madison, WI), Petersen; Michael W. (Sauk City, WI), Gilbertson; Larry (Chesterfield, MO), Walters; David (North Stonington, CT), Johnson; Susan (New London, CT), Huang; Shihshieh (Stonington, CT), Chomet; Paul S. (Mystic, CT)
Assignee:	Monsanto Technology LLC (St. Louis, MO)
Application Number:	13/558,336
Patent Claims:	1. A method of preparing marker-free seeds from a transgenic plant comprising the steps of: a) obtaining seeds from a transgenic plant, the plant comprising a first DNA segment comprising a nucleic acid of interest and a second DNA segment comprising a marker gene physically linked to a DNA cassette that is operably linked to a promoter functional in said seed, wherein the DNA cassette confers a detectable phenotype; b) screening the seeds for the absence of the detectable phenotype; and c) selecting at least a first seed that lacks the detectable phenotype to obtain a seed free of the marker gene; wherein the DNA cassette comprises a DNA sequence encoding an antisense or sense RNA that silences an endogenous gene to result in the detectable phenotype. 2. The method of claim 1, wherein step c) further comprises assaying the seed for the presence of the nucleic acid of interest and selecting a seed that comprises the nucleic acid of interest. 3. The method of claim 1, wherein the marker gene is a selectable, or screenable marker gene. 4. The method of claim 3, wherein the DNA cassette encodes an RNA that is translationally or transcriptionally fused to the selectable marker gene. 5. The method of claim 1, wherein the DNA cassette comprises a pair of inverted repeats of a DNA fragment homologous to the endogenous gene. 6. The method of claim 5, wherein the inverted DNA fragment repeat is embedded in an intron within the marker gene. 7. The method of claim 1, wherein the first DNA segment and second DNA segment overlap. 8. The method of claim 1, wherein the seed selected in step c) lacks the marker gene and DNA cassette. 9. The method of claim 1, wherein the transgenic plant or a progenitor thereof of any previous generation was co-transformed with the first and second DNA segments on separate DNA constructs. 10. The method of claim 1, wherein the first and second DNA segments are bounded by different T-DNA border sequences. 11. The method of claim 10, wherein step a) comprises transforming the transgenic plant or a progenitor thereof of any previous generation with a single DNA construct comprising the first and second DNA segments. 12. The method of claim 1, wherein the transgenic plant was produced by transforming the plant or a progenitor thereof of any previous generation with a DNA construct comprising (i) the first DNA segment flanked by left and right T-DNA borders comprising a nucleic acid of interest, and (ii) the second DNA segment flanked by a second set of left and right T-DNA borders, wherein the second DNA segment further comprises a marker gene operably linked to a promoter functional in the transgenic plant. 13. The method of claim 1, wherein the first and second DNA segments are not genetically linked in said transgenic plant. 14. The method of claim 1, wherein the first and second DNA segments are genetically linked in said transgenic plant. 15. The method of claim 1, wherein the transgenic plant was produced by introducing the first and second DNA segments into said plant or a progenitor thereof of any previous generation by transformation mediated by a bacterial strain selected from the group consisting of Agrobacterium, Rhizobium, Mesorhizobium, and Sinorhiboziumor; or by microprojectile bombardment. 16. The method of claim 3, wherein the selectable marker gene encodes a product selected from the group consisting of CP4 EPSPS, phosphinothricin acetyltransferase, DMO, NptII, glyphosate acetyl transferase, mutant acetolactate synthase, methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox, hygromycin phosphotransferase and 5-methyl tryptophan resistant anthranilate synthase. 17. The method of claim 1, wherein the DNA cassette comprising a DNA sequence encoding the antisense or sense RNA comprises a DNA fragment with at least 19 or 21 bp of homology to the endogenous gene, or wherein said DNA cassette comprises a pair of inverted repeats of said DNA fragment; wherein said endogenous gene is selected from the group consisting of an anthocyanin synthesis gene, OsCDPK2, AP2, ARF2, ANT transcription factor, LEC2, Snf-1, Kas4, zein [inverted repeats], and B-peru. 18. The method of claim 1, wherein the DNA cassette is operably linked to a promoter functional in a tissue selected from an embryo, seed endosperm, cotyledon, aleurone, and seed coat. 19. The method of claim 1, wherein the DNA cassette comprises a nucleic acid sequence operably linked to a promoter selected from the group consisting of a napin promoter, a beta-phaseolin promoter, a beta-conglycinin subunit promoter, a zein promoter, a rice glutelin (Osgt-1) promoter, an oleosin promoter, a starch synthase promoter, a globulin 1 promoter, a barley-aleurone-specific lipid transfer protein (LTP2) promoter, an alpha-amylase promoter, a chitinase promoter, a beta-glucanase promoter, a cysteine proteinase promoter, a glutaredoxin promoter, a barley late embryogenesis-abundant protein (HVA1) promoter, a serine carboxypeptidase II promoter, a catalase promoter, an alpha-glucosidase promoter, a beta-amylase promoter, a maize viviparous-1 (VP1) promoter, a Vicia faba unknown seed protein (USP) promoter, a Vicia faba unknown seed protein (USP88) promoter, a Vicia faba unknown seed protein (USP99) promoter, a Lectin promoter, and a bronze2 promoter. 20. The method of claim 1, wherein the detectable phenotype is selected from the group consisting of seed color, seed opacity, seed germinability, seed viability, seed shape and seed texture. 21. The method of claim 1, wherein the detectable phenotype is assayed by detection of a catalytic activity. 22. The method of claim 1, wherein the detectable phenotype is a tissue ablation phenotype. 23. The method of claim 1, wherein the detectable phenotype results in a defective or aborted seed. 24. The method of claim 1, wherein step c) is carried out by an automated seed sorting machine. 25. A DNA construct comprising (a) a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants, and (b) a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a seed operably linked to a DNA cassette that confers a detectable phenotype in seeds that comprise the DNA cassette and a marker gene operably linked to a promoter functional in plants; wherein the DNA cassette that confers a detectable phenotype comprises a DNA sequence encoding an antisense or sense RNA that silences an endogenous gene to result in the detectable phenotype. 26. The DNA construct of claim 25, wherein the marker gene is a selectable marker gene. 27. The construct of claim 25, wherein the gene of interest confers a trait selected from the group consisting of herbicide tolerance, insect or pest resistance, disease resistance, increased biomass, modified fatty acid metabolism, modified carbohydrate metabolism, and modified nutritional quality. 28. The construct of claim 25, wherein the DNA cassette and selectable marker gene are operably linked to the same promoter. 29. The construct of claim 25, wherein the DNA cassette and the selectable marker gene are operably linked to different promoters. 30. The construct of claim 25, wherein the selectable marker gene encodes a product selected from the group consisting of CP4 EPSPS, phosphinothricin acetyltransferase, DMO, NptII, glyphosate acetyl transferase, mutant acetolactate synthase, methotrexate resistant DHFR, dalapon dehalogenase, PMI, Protox, hygromycin phosphotransferase and 5-methyl tryptophan resistant anthranilate synthase. 31. The construct of claim 25, wherein the DNA cassette comprising a DNA sequence encoding the antisense or sense RNA comprises a DNA fragment with at least 19 or 21 bp of homology to the endogenous gene [or an inverted repeat sequence], or wherein said DNA cassette comprises a pair of inverted repeats of said DNA fragment; wherein said endogenous gene is selected from the group consisting of an anthocyanin synthesis gene, OsCDPK2, AP2, ARF2, ANT transcription factor, LEC2, Snf-1, Kas4, zein [inverted repeats], and B-peru. 32. The construct of claim 25, wherein the DNA cassette is operably linked to a promoter functional in a tissue selected from the group consisting of an embryo, seed endosperm, cotyledon, aleurone, and seed coat. 33. The construct of claim 25, wherein the DNA cassette is operably linked to a promoter selected from the group consisting of: a napin promoter, a beta-phaseolin promoter, a beta-conglycinin subunit promoter, a zein promoter, a rice glutelin (Osgt-1) promoter, an oleosin promoter, a starch synthase promoter, a globulin 1 promoter, a barley aleurone-specific lipid transfer protein (LTP2) promoter, an alpha-amylase promoter, a chitinase promoter, a beta-glucanase promoter, a cysteine proteinase promoter, a glutaredoxin promoter, a barley late embryogenesis-abundant protein (HVA1) promoter, a serine carboxypeptidase II promoter, a catalase promoter, an alpha-glucosidase promoter, a beta-amylase promoter, a maize viviparous-1 (VP1) promoter, a Vicia faba unknown seed protein (USP) promoter, a Vicia faba unknown seed protein (USP88) promoter, a Vicia faba unknown seed protein (USP99) promoter, a Lectin promoter, and a bronze2 promoter. 34. A transgenic cell transformed with the construct of claim 25. 35. A transgenic plant transformed with the construct of claim 25. 36. A transgenic plant co-transformed with a DNA construct comprising a first DNA segment comprising left and right T-DNA borders flanking a gene of interest operably linked to a promoter functional in plants, and a second DNA construct containing a second DNA segment comprising a second set of left and right T-DNA borders flanking a promoter functional in a seed operably linked to a DNA cassette encoding an antisense or sense RNA that silences an endogenous gene and that confers a detectable phenotype in seeds that comprise the DNA cassette and a marker gene operably linked to a promoter functional in plants. 37. A cell of the plant of claim 36. 38. A DNA construct comprising right and left T-DNA borders, wherein a first DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located downstream in the 5' to 3' direction from a right border; and a second DNA segment comprising a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette and a marker gene operably linked to a promoter functional in plants is located downstream in the 5' to 3' direction from a left border; wherein the DNA cassette that confers a detectable phenotype comprises DNA encoding an antisense or sense RNA that silences an endogenous gene to result in the detectable phenotype. 39. A DNA construct comprising right and left T-DNA borders, wherein a first DNA segment comprising a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette and a marker gene operably linked to a promoter functional in plants is located downstream in the 5' to 3' direction from a right border, and a second DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located downstream in the 5' to 3' direction from a left border; wherein the DNA cassette that confers a detectable phenotype comprises DNA encoding an antisense or sense RNA that silences an endogenous gene to result in the detectable phenotype. 40. A DNA construct comprising in the 5' to 3' direction first and second right T-DNA borders, wherein a first DNA segment comprising a gene of interest operably linked to a promoter functional in plants is located downstream in the 5' to 3' direction from the first right border, and a second DNA segment comprising a DNA cassette that confers a detectable phenotype to plant seeds that comprise the DNA cassette and a marker gene operably linked to a promoter functional in plants located downstream in the 5' to 3' direction from the second right border; wherein the DNA cassette that confers a detectable phenotype comprises DNA encoding an antisense or sense RNA that silences an endogenous gene to result in the detectable phenotype.

Details for Patent 8,829,275

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2026-05-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2026-05-12
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2026-05-12