Claims for Patent: 7,267,979

Summary for Patent: 7,267,979

Title:	Method of controlling gene silencing using site specific recombination
Abstract:	This invention relates to methods of controlling gene silencing using site-specific recombination. A variety of constructs are provided which are useful for conditional or regulated gene silencing in plants, comprising a suite of constitutive, inducible, tissue-specific or developmental stage-specific promoters operably linked to target sequences (TS). Recombinase inversion or excision yields double-stranded TS RNA, which thereby functions to trigger endogenous gene silencing mechanisms. By matching promoters, responsive to various inducers, plant tissues or plant developmental states with the recombinase systems, transcriptional stop fragments or introns and target sequences, gene silencing of virtually any target sequence may be modulated at any plant development stage or in any plant generation. This is especially useful, when genes responsible for gene silencing are down-regulated to permit expression of particular transgenes at levels greater than permitted when gene silencing is activated.
Inventor(s):	Yadav; Narendra S. (Chadds Ford, PA)
Assignee:	Pioneer Hi-Bred International, Inc. (Johnston, IA)
Application Number:	10/611,748
Patent Claims:	1. A gene silencing site-specific recombination system comprising: a) a first recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3' region; and b) a second recombinase element having the general structure: RS-X-RS*-Y wherein: i) RS and RS* are opposingly oriented recombinase sites responsive to the recombinase; ii) X is a nucleic acid fragment comprising at least one second promoter in a 3' to 5' orientation, wherein X comprises 5' Intron-TSINV-P2INV; and iii) Y is a nucleic acid fragment comprising 3' Intron-TSINV-polyA; wherein: 1) P2INV comprises an inverted second promoter whose orientation is from 3'-5'; 2) TSINV is an inverted target sequence whose orientation is from 3'-5', and is complementary to a sequence in the coding region of a target gene; 3) polyA is the 3' region of a gene; 4) 5' Intron is the N-terminal portion of an intron; and, 5) 3' Intron is the C-terminal portion of said intron; wherein, when said system is present in a plant cell comprising the target gene, expression of the recombinase results in inversion of X, yielding a recombined second recombinase element, RS-P2-TS-5' Intron-RS*-3' Intron-TSINV-polyA, wherein transcription of TS and TSINV by P2 results in the production of double-stranded RNA that causes silencing of the target gene. 2. The gene silencing site-specific recombination system according to claim 1 wherein the recombinase and recombinase site are selected from the group consisting of Cre-lox, FLP/FRT, R/RS, and Gin/gix. 3. The gene silencing site-specific recombination system according to claim 1 wherein the first promoter and at least one second promoter are selected from the group consisting of a) constitutive plant promoters; b) plant tissue-specific promoters; c) plant development stage-specific promoters; d) chemically-inducible plant promoters; and e) viral promoters. 4. A gene silencing site-specific recombination system comprising: a) a first recombinase element having the general structure P1-R, wherein P1 is a first promoter and R is a recombinase coding sequence and 3' region; and b) a second recombinase element having the general structure RS-5' Intron-TSINV-P2INV-RS*-3' Intron-TSINV-polyA, wherein: i) RS and RS* are opposingly oriented recombinase sites responsive to the recombinase; ii) 5' Intron is the N-terminal portion of an intron; iii) TSINV is an inverted target sequence and whose orientation is from 3'-5', and is complementary to a sequence in the coding region of a target gene; iv) P2INV is an inverted second promoter whose orientation is from 3'-5'; v) 3'Intron is the C-terminal portion of said intron; and vi) polyA is the 3' region of a gene; wherein, when the system is present in a plant cell comprising the target gene, recombinase expression results in recombination of the second recombinase element to yield RS-P2-TS-5' Intron-RS*-3' Intron-TSINV-polyA, wherein P1 and P2 are operably linked to their down stream elements, wherein following transcription of the recombined second recombinase element, the intron is excised by mRNA splicing and the transcripts of TS and TSINV hybridize to form a double-stranded RNA that causes silencing of the target gene. 5. The gene silencing site-specific recombination system according to claim 4 wherein the first promoter is a germline promoter. 6. The gene silencing site-specific recombination system according to claim 5 wherein the germline promoter is selected from the group consisting of: male germline-specific promoters; female germline-specific promoters; common germline-specific promoters; floral common germline-specific promoters; vegetative shoot apical meristem-specific promoters; and floral shoot apical meristem-specific promoters. 7. The gene silencing site-specific recombination system according to claim 6 wherein the male germline-specific promoter is derived from genes selected from the group consisting of genes specific to anther primordia, anther sporophyte and pollen gametophyte. 8. The gene silencing site-specific recombination system according to claim 6 wherein the common germline-specific promoter is derived from genes selected from the group consisting of Apetala 3 (AP3), Pistillata (Pl), synthetic anther promoter, TA29, BCP1 and orthologs thereof. 9. The gene silencing site-specific recombination system according to claim 6 wherein the common germline-specific promoter is derived from genes selected from the group consisting of Leafy (LFY), Apetala 3 (AP3), Pistillata (Pl), Apetala 1 (AP1), Agamous (AG), Pistillata (Pl) and orthologs thereof. 10. The gene silencing site-specific recombination system according to claim 6 wherein the floral common germline-specific promoter is derived from genes selected from the group consisting of Agamous (AG), Apetala 1 (AP1), Apetala 3 (AP3), Leafy (LFY) and orthologs thereof. 11. The gene silencing site-specific recombination system according to claim 6 wherein the vegetative shoot apical meristem-specific promoter is selected from the group consisting of Agamous (AG), Apetala 1 (AP1), Apetala 3 (AP3), Leafy (LFY), Aintegumenta (ANT), Clavata 3 (CLV3), Wushel (WUS), Meristemless (STM) and orthologs thereof. 12. The gene silencing site-specific recombination system according to claim 4 wherein the second promoter is selected from the group consisting of: a) constitutive plant promoters; b) plant tissue-specific promoters; c) plant development stage-specific promoters; d) chemically-inducible plant promoters; and e) viral promoters. 13. The gene silencing site-specific recombination system according to claim 1 or claim 4 wherein the target sequence silences a target gene selected from the group consisting of: a) a gene encoding an enzyme of a biosynthetic pathway; b) a gene encoding a storage protein; c) a gene conveying sterility; d) a gene conveying a specific phenotype on a plant or plant cell; e) a hormone biosynthetic gene; and f) a gene involved in gene silencing. 14. The gene silencing site-specific recombination system according to claim 13 wherein the genes involved in gene silencing are selected from the group consisting of: a) qde-1, qde-2, and qde-3; b) rde-1, rde-2, rde-3, and rde-4; c) mut-2, and mut-7; d) ego-1; e) AGO1; f) SGS-2/SDE-1, SGS-1, and SGS-3; g) RdRP; and, h) Dicer. 15. The gene silencing site-specific recombination system according to claim 4 wherein the recombinase coding sequences and recombinase site are selected from the group consisting of Cre-lox, FLP/FRT, R/RS, and Gin/gix. 16. The gene silencing site-specific recombination system according to claim 4 wherein the first recombinase element and the second recombinase element may be genetically linked or unlinked. 17. The gene silencing site-specific recombination system according to claim 16 wherein the first recombinase element and the second recombinase element are genetically unlinked.

Details for Patent 7,267,979

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		2022-07-01
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2022-07-01
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2022-07-01