Claims for Patent: 7,157,272

Summary for Patent: 7,157,272

Title:	Constructs for gene expression analysis
Abstract:	The present invention relates generally to constructs and their use in gene expression or gene regulation assays. More particularly, the present invention provides expression vectors and/or reporter vectors providing kinetics of protein expression with improved temporal correlation to promoter activity. Even more particularly, the invention provides expression vectors comprising a transcribable polynucleotide which comprises a sequence of nucleotides encoding a RNA element that modulates the stability of a transcript corresponding to the transcribable polynucleotide. The present invention provides, inter alia, novel vectors, useful for identifying and analysing cis- and trans-acting regulatory sequences/factors as well as vectors and genetically modified cell lines or organisms that are particularly useful for drug screening and drug discovery.
Inventor(s):	Daly; John (City Beach, AU)
Assignee:	Gene Stream Pty Ltd. (City Beach, AU)
Application Number:	10/658,093
Patent Claims:	1. A recombinant construct comprising in operable linkage: a polynucleotide that encodes a polypeptide comprising a protein-destabilizing element, and a nucleic acid sequence that encodes an RNA destabilizing element that reduces the stability of a transcript encoded by the polynucleotide in a eukaryotic cell, wherein the polynucleotide and the nucleic acid sequence are heterologous to each other. 2. The construct according to claim 1, wherein the protein-destabilizing element is selected from the group consisting of: a PEST sequence, an N-terminal destabilizing amino acid and a ubiquitin. 3. The construct according to claim 1, wherein the polypeptide is a reporter protein. 4. The construct according to claim 3, wherein the reporter protein is an enzymatic protein or a protein associated with the emission of light. 5. The construct according to claim 3, wherein the reporter protein is a fluorescent protein or a luminescent protein. 6. The construct according to claim 3, wherein the reporter protein is selected from the group consisting of: Luciferase, Green Fluorescent Protein, Red Fluorescent Protein, SEAP and CAT. 7. The construct according to claim 1, further comprising a cloning site for introducing a sequence of nucleotides in operable connection with the polynucleotide and the nucleic acid sequence. 8. The construct according to claim 7, wherein the cloning site is a multiple cloning site. 9. The construct according to claim 7, wherein the sequence of nucleotides comprises a transcriptional control element. 10. The construct according to claim 7, wherein the sequence of nucleotides comprises a promoter. 11. The construct according to claim 7, wherein the sequence of nucleotides comprises a cis-acting regulatory element. 12. The construct according to claim 11, wherein the cis-acting regulatory element is selected from the group consisting of: an enhancer of transcription, an enhancer of translation, an enhancer of mRNA splicing, an enhancer of mRNA export, an enhancer of mRNA degradation, a repressor of transcription, a repressor of translation, a repressor of mRNA splicing, a repressor of mRNA export and a repressor of mRNA degradation. 13. The construct according to claim 1, further comprising a polyadenylation sequence. 14. The construct according to claim 1, further comprising a selectable marker. 15. The construct according to claim 1, further comprising an origin of replication. 16. The construct according to claim 1, further comprising a translational enhancer. 17. The construct according to claim 1, which is a vector. 18. The construct according to claim 1, further comprising one or more members selected from the group consisting of: a multiple cloning site for introducing a sequence of nucleotides; a reporter gene; a transcriptional enhancer for enhancing transcription of the polynucleotide; a translational enhancer for enhancing translation of the transcript encoded by the polynucleotide; a polyadenylation sequence; a selectable marker gene; an origin of replication; an intron; and a mRNA nuclear export signal. 19. The construct according to claim 7 or claim 18, further comprising at least one site which is cleavable enzymatically, chemically or otherwise to provide a linearised vector into which PCR amplification products can be directly inserted. 20. The construct according to claim 1, wherein the nucleic acid sequence is from a gene selected from the group consisting of: c-fos, c-jun, c-myc, GM-CSF, IL-3, TNF-alpha, IL-2, IL-6, IL-8, IL-10, Urokinase, bcl-2, SGLT1 (Na(+j-coupled glucose transporter), Cox-2 (cyclooxygenase 2), IL-8, PAI-2 (plasminogen activator inhibitor type 2), beta1-adrenergic receptor and GAP43. 21. The construct according to claim 1, wherein the nucleic acid sequence is SEQ ID NO:19. 22. The construct according to claim 1, wherein the polypeptide is a protein having at least a light-emitting activity and a selection marker activity. 23. The construct according to claim 22, wherein the polypeptide is encoded by a chimeric gene comprising a coding sequence from a gene encoding a light-emitting protein and a coding sequence from a gene encoding a selectable marker protein. 24. The construct according to claim 22, wherein the polypeptide is encoded by a chimeric gene comprising a coding sequence from a gene encoding: a light-emitting protein selected from the group consisting of: Green Fluorescent Protein, Luciferase; and a coding sequence from a gene encoding a selectable marker protein selected from the group consisting of: kanamycin kinase, neomycin phosphotransferase, aminoglycoside phosphotransferase, puromycin N-acetyl transferase, and puromycin resistance protein. 25. An isolated or recombinant cell comprising the construct according to claim 1. 26. The cell according to claim 25, wherein the cell is a eukaryotic cell. 27. The cell according to claim 25, wherein the cell is a mammalian cell. 28. The cell according to claim 25, wherein the cell is a human cell. 29. The cell according to claim 25, wherein the cell is a plant cell. 30. The construct according to claim 1, wherein the RNA destabilizing element comprises an AU-rich element. 31. The construct according to claim 30, wherein the AU-rich element comprises the sequence set forth in SEQ ID NO:1. 32. The construct according to claim 1, wherein the polypeptide is a reporter protein comprising a PEST sequence. 33. The construct according to claim 32, wherein the reporter protein comprises Luciferase. 34. The construct according to claim 32, wherein the reporter protein comprises firefly luciferase. 35. The construct according to claim 32, wherein the reporter protein comprises Renilla luciferase. 36. The construct according to claim 1, wherein the RNA destabilizing element comprises an AU-rich element and wherein the polypeptide is a reporter protein that comprises firefly luciferase and a PEST sequence. 37. The construct according to claim 1, wherein the RNA destabilizing element comprises an AU-rich element and wherein the polypeptide is a reporter protein that comprises Renilla luciferase and a PEST sequence.

Details for Patent 7,157,272

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2021-03-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2021-03-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2021-03-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2021-03-09