Claims for Patent: 5,043,270
✉ Email this page to a colleague
Summary for Patent: 5,043,270
Title: | Intronic overexpression vectors |
Abstract: | DNA constructs are provided employing intronically positioned expression, systems, where one of the genes is a dominant gene, usually amplifiable, and the other gene encodes a sequence of interest. Higher levels of co-expression are achieved than when the genese are ligated in tandem. Specifically, the gene of interest is inserted into the intron of a DHFR minigene, the construct transformed into a mammalian cell and the resulting transformants stressed with progressively increasing levels of methotrexate. Substantially increasing levels of co-expression are achieved with increasing levels of methotrexate. |
Inventor(s): | Abrams; John M. (New York, NY), Schimke; Robert T. (Palo Alto, CA), Thorpe; Susan M. (Copenhagen, DK) |
Assignee: | The Board of Trustees of the Leland Stanford Junior University (Stanford, CA) |
Application Number: | 07/331,434 |
Patent Claims: | 1. A non-naturally occurring DNA construct, said DNA construct comprising an external genetic element and an internal genetic element, said external genetic element
comprising a primary gene, primary transcriptional initiation and termination sites, and primary transcriptional regulatory region regulating transcription of said primary gene, said primary gene containing at least one intron, said internal genetic
element comprising a secondary gene, secondary transcriptional initiation and termination sites, and secondary transcriptional regulatory region regulating transcription of said secondary gene, said internal genetic element being within said at least one
intron, with at least one member of the group composed of said primary gene and said secondary gene being a gene capable of selection.
2. A DNA construct of claim 1, wherein said primary gene is said gene capable of selection. 3. A DNA construct of claim 1, wherein said gene capable of selection is an amplifiable gene capable of amplification by means of a selective agent. 4. A DNA construct according to claim 3, wherein said amplifiable gene is DHFR. 5. A vector comprising a DNA construct according to claim 4. 6. A vector according to claim 5, wherein said vector is capable of replication in a prokaryotic host. 7. A vector according to claim 5, wherein said vector is capable of replication in a eukaryotic host. 8. A DNA construct of claim 1, wherein said intron is bounded by coding regions of said gene capable of selection. 9. A DNA construct of according to claim 1, wherein said secondary gene is an open reading frame capable of selection. 10. A DNA construct according to claim 1, wherein said secondary gene is an anti-sense sequence of a gene in a cellular host in which said primary and secondary transcriptional regulatory regions are functional. 11. A vector comprising a DNA construct according to claim 1. 12. A vector according to claim 11, wherein said vector is capable of replication in a prokaryotic host. 13. A vector according to claim 11, wherein said vector is capable of replication in a eukaryotic host. 14. Eukaryotic host cells in culture according to claim 1, wherein said secondary gene is an open reading frame capable of expression. 15. A method for producing a protein of interest, employing eukaryotic cells containing multiple copies of a DNA construct according to claim 1, wherein one member of said group is an amplifiable gene capable of amplification by means of a selective agent, and the other member of said group is a gene encoding the protein of interest, said method comprising: growing said cells in a nutrient medium, whereby said protein is expressed; and harvesting said protein. 16. A method according to claim 15, wherein said nutrient medium is selective for said amplifiable gene. 17. Eukaryotic host cells in culture, said cells comprising a DNA construct according to claim 1, said DNA construct being integrated into a chromosome of said host cells. 18. Eukaryotic host cells in culture according to claim 17, wherein said primary gene is said gene capable of selection. 19. Eukaryotic host cells in culture according to claim 17, wherein said gene capable of selection is an amplifiable gene capable of amplification by means of a selective agent. 20. Eukaryotic host cells in culture according to claim 19, wherein said amplifiable gene is DHFR. 21. Eukaryotic host cells in culture according to claim 17, wherein said intron is bounded by coding regions of said gene capable of selection. 22. Eukaryotic host cells in culture according to claim 17, wherein said secondary gene is an anti-sense sequence of a gene in a cellular host in which said primary and secondary transcriptional regulatory regions are functional. 23. Eukaryotic host cells in culture according to claim 17, wherein said external and internal genetic elements are present in multiple tandem copies. 24. Eukaryotic host cells in culture according to claim 17, wherein said external and internal genetic elements are present in multiple tandem copies. 25. A method for producing eukaryotic cells having multiple copies of a sequence of interest, wherein said cell comprises introducing into said cells a DNA construct comprising two genetic elements, a first external genetic element comprising first transcriptional initiation and termination regulatory regions for transcription and a first gene comprising an intron under the transcriptional regulation of said first regulatory regions; and a second internal genetic element within said intron comprising second transcriptional initiation and termination regulatory regions and a second gene under the transcriptional regulation of said second regulatory regions, with the proviso that one of said genetic elements is a gene capable of amplification, said method comprising: growing said cells in a selective medium comprising an amplifying agent for sufficient time for amplification to occur; and selecting cells having multiple copies of said sequence of interest. 26. A method according to claim 25, wherein said amplifying agent is sequentially raised in concentration. |
Details for Patent 5,043,270
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 | ⤷ Try a Trial | 2039-02-26 |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | For Injection | 103132 | ⤷ Try a Trial | 2039-02-26 | |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | Injection | 103132 | ⤷ Try a Trial | 2039-02-26 | |
>Applicant | >Tradename | >Biologic Ingredient | >Dosage Form | >BLA | >Approval Date | >Patent No. | >Expiredate |
Make Better Decisions: Try a trial or see plans & pricing
Drugs may be covered by multiple patents or regulatory protections. All trademarks and applicant names are the property of their respective owners or licensors. Although great care is taken in the proper and correct provision of this service, thinkBiotech LLC does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free. thinkBiotech performs no independent verification of facts as provided by public sources nor are attempts made to provide legal or investing advice. Any reliance on data provided herein is done solely at the discretion of the user. Users of this service are advised to seek professional advice and independent confirmation before considering acting on any of the provided information. thinkBiotech LLC reserves the right to amend, extend or withdraw any part or all of the offered service without notice.