Claims for Patent: 6,452,067

Summary for Patent: 6,452,067

Title:	Methods to assay for post-transcriptional suppression of gene expression
Abstract:	This invention provides methods for identifying plant cells that exhibit post-transcriptional gene silencing (PTGS) of a chosen gene. The methods involve the use of suppression-sensitive reporter genes that, when introduced into plant cells, are expressed at a lower level in cells that exhibit PTGS than in cells that are not silenced for the particular gene.
Inventor(s):	Bedbrook; John R. (Piedmont, CA), Gutterson; Neal (Oakland, CA), Oeller; Paul W. (Berkley, CA)
Assignee:	DNA Plant Technology Corporation (Oakland, CA)
Application Number:	09/156,210
Patent Claims:	1. A method for detecting post-transcriptional silencing of a chosen gene in a plant cell, the method comprising: introducing into the plant a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence wherein such targeting nucleotide sequence is at least 80% identical to the chosen gene, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and determining the level of expression of the reporter coding sequence to determine whether post-transcriptional gene silencing of the chosen gene has occurred. 2. The method of claim 1, wherein the chosen gene is an endogenous gene. 3. The method of claim 2, wherein the plant cell comprises a transgene which produces a transcript that is at least 80% identical to the endogenous gene. 4. The method of claim 1, wherein the chosen gene is a transgene. 5. The method of claim 4, wherein the targeting nucleotide sequence is at least 95% identical to a nucleotide sequence present in a plant virus. 6. The method of claim 1, wherein the post-transcriptional gene silencing is selected from the group consisting of sense suppression and antisense suppression. 7. The method of claim 1, wherein the plant cell is contained in a plant. 8. The method of claim 1, wherein the promoter is a constitutive promoter. 9. The method of claim 8, wherein the promoter is a cauliflower mosaic virus 35S promoter or a figwort mosaic virus promoter. 10. The method of claim 8, wherein the promoter is a heterologous promoter with respect to the targeting nucleotide sequence. 11. The method of claim 1, wherein the introducing is by cocultivating the plant cell with Agrobacterium cells that comprise a nucleic acid which comprises the SSR gene flanked by functional T-DNA borders. 12. The method of claim 11, further comprising, prior to said introducing step, excising a plant cell from a plant. 13. The method of claim 12, further comprising, after said excising step, preculturing said cell on artificial medium, wherein said cell is contained in a plant tissue. 14. The method of claim 1, wherein the SSR gene is incapable of expression in bacterial cells. 15. The method of claim 14, wherein the SSR gene further comprises an intron. 16. The method of claim 14, wherein the SSR gene further comprises a bacterial transcription termination signal at the 3' end of the targeting nucleotide sequence. 17. The method of claim 1, wherein the targeting nucleotide sequence is located in a 3' untranslated region of the reporter coding sequence. 18. The method of claim 1, wherein the reporter coding sequence encodes a reporter moiety selected from the group consisting of .beta.-glucuronidase (uidA), luciferase, green fluorescent protein (GFP), and chloramphenicol acetyltransferase (CAT). 19. The method of claim 18, wherein the reporter moiety is an Escherichia coli .beta.-glucuronidase. 20. The method of claim 1, wherein the expression of the SSR gene is detected quantitatively. 21. The method of claim 1, wherein the method further comprises introducing into the plant cell a non-suppression reporter (NSR) gene which comprises a second reporter coding sequence which is different from the reporter coding sequence included in the SSR gene and lacks a targeting nucleotide sequence, and determining the level of expression of both the reporter coding sequence and the non-suppression sensitive reporter to determine whether post-transcriptional gene silencing of the chosen gene has occurred. 22. The method of claim 21, wherein the second reporter coding sequence encodes a reporter moiety selected from the group consisting of .beta.-glucuronidase (uidA), luciferase, green fluorescent protein (GFP), and chloramphenicol acetyl transferase (CAT). 23. The method of claim 21, wherein the introducing is by cocultivating the plant cell with Agrobacterium cells that comprise a nucleic acid which comprises the NSR gene flanked by functional T-DNA borders. 24. The method of claim 23, wherein the SSR gene is flanked by the same functional T-DNA borders as is the NSR gene. 25. The method of claim 23, wherein the SSR gene and the NSR gene are flanked by two separate sets of functional T-DNA borders. 26. The method of claim 25, wherein each of the two sets of function T-DNA borders is present in a single Agrobacterium strain. 27. A method for detecting transgene-induced post-transcriptional silencing of an endogenous gene in a plant cell, the method comprising: introducing into the plant cell a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence wherein such targeting nucleotide sequence is at least 80% identical to the endogenous gene, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and determining the level of expression of the reporter coding sequence to determine whether post-transcriptional silencing of the endogenous gene has occurred. 28. A method for assaying a plant to determine whether a chosen gene is post-transcriptionally silenced, the method comprising: introducing into cells obtained from the plant a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence that is at least 80% identical to the chosen gene, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and determining the level of expression of the reporter coding sequence gene to determine whether post-transcriptional silencing of the chosen gene has occurred. 29. The method of claim 28, wherein the cells comprise a transgene that is at least 95% identical to the chosen gene. 30. The method of claim 28, wherein the method further comprises introducing into the cells a non-suppression sensitive reporter (NSR) gene which comprises a reporter gene that encodes a reporter moiety which is different from the reporter moiety encoded by the SSR gene, and determining the level of expression of both the SSR gene and the NSR gene, wherein a decreased level of SSR gene expression relative to NSR gene expression is indicative of post-transcriptional gene silencing. 31. A method of identifying plant cells that use post-transcriptional gene silencing to resist infection by plant virus, the method comprising: introducing into the plant cells a nucleic acid comprising a promoter operably linked to a suppression-sensitive reporter (SSR) gene which comprises a) a reporter coding sequence, and b) a targeting nucleotide sequence wherein such targeting nucleotide sequence is at least 80% identical to a nucleotide sequence obtained from the plant virus, wherein the reporter coding sequence and the targeting nucleotide sequence are transcribed as a single mRNA transcript; and determining the level of expression of the reporter coding sequence, wherein a reduction in reporter coding sequence expression is indicative of cells that are resistant to infection by the plant virus. 32. The method of claim 31, wherein the plant virus is selected from the group consisting of potato virus Y and related potyviruses, tobacco etch virus, and tobacco vein mottling virus.

Details for Patent 6,452,067

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		2017-09-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2017-09-19
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2017-09-19