Claims
- 1. An expression vector which comprises:
- a first DNA sequence encoding a DHFR protein; and
- a second DNA sequence encoding human tPA;
- wherein each of said first and second sequences is operably linked to a DNA sequence capable of effecting its expression.
- 2. The vector of claim 1 wherein the DHFR protein is wild type DHFR.
- 3. The vector of claim 2 which is effective in cells deficient in DHFR.
- 4. The vector of claim 1 wherein in the DHFR protein has a low binding affinity for MTX.
- 5. The vector of claim 1 which is effective in cells sensitive to MTX.
- 6. The vector capable of effecting expression of a DNA sequence encoding human tPA and of a DNA sequence encoding a DHFR protein, said sequences being contained therein, in cells transformed by said vector.
- 7. The plasmid pETPFR.
- 8. Cells transformed by the vector which comprises: a first DNA sequence encoding a DHFR protein; and a second DNA sequence encoding human tPA; wherein each of said first and second sequences is operably linked to a DNA sequence capable of affecting its expression.
- 9. Cells transformed by the vector pETPFR.
- 10. A method of amplifying the DNA sequence encoding tPA and increasing the production of tPA in a host cell culture which method comprises transfecting the host cells with an expression vector which comprises: a first DNA sequence encoding a DHFR protein; and a second DNA sequence encoding human tPA; wherein each of said first and second sequences is operably linked to a DNA sequence capable of effecting its expression, and growing the cells in the presence of an effective amplifying concentration of MTX.
- 11. The method according to claim 10 wherein said tPA production is in amounts greater than 20 pg per cell per day.
Parent Case Info
This is a Continuation application of Ser. No. 07/149,990, filed Jan. 27, 1988, now abandoned, which is a continuing application under 35 U.S.C. 120/121 of U.S. Ser. No. 06/459,153, filed Jan. 19, 1983, now abandoned. Reference is hereby made under 35 U.S.C. 120/121 to U.S. Ser. No. 06/374,860, filed May 5, 1982, U.S. Ser. No. 06/398,003, filed July 14, 1982, both now abandoned, and U.S. Ser. No. 06/483,052, filed Apr. 7, 1983, now U.S. Pat. No. 4,877,075.
US Referenced Citations (2)
Number |
Name |
Date |
Kind |
4399216 |
Axel et al. |
Aug 1983 |
|
4419446 |
Howley et al. |
Dec 1983 |
|
Foreign Referenced Citations (5)
Number |
Date |
Country |
0037687 |
Oct 1981 |
EPX |
0054223 |
Jun 1982 |
EPX |
0093619 |
Nov 1983 |
EPX |
0117060 |
Aug 1984 |
EPX |
8102425 |
Sep 1981 |
WOX |
Non-Patent Literature Citations (8)
Entry |
Odenakker et al., "Messenger RNA for Human Tissue Plasminogen Activator", Eur. J. Biochem. 121: 269 (1982). |
Haber et al., "Unstable Amplification of an Altered Dihydrofolate Reductase Gene Associated with Double-Minute Chromosomes", Cell 26: 355 (1981). |
Chang et al., "Phenotypic Expression in E. coli of a DNA Sequence Coding for Mouse Dihydrofolate Reductase", Nature 275: 617 (1978). |
Blattner et al.; "Cloning Human Fetal Gamma-Globin and Mouse Alpha-Type Globin DNA: Preparation and Screening of Shotgun Collections", Science 202: 334 (1978). |
Christman, Judith K. et al., Proc. Natl. Acad. Sci. 79: 1815 (1982). |
Kaufman, Randal J. et al., J. Mol. Biol. 159: 601 (1982). |
Ringold, Gordon et al., Journal of Molecular and Applied Genetics 1: 165 (1981). |
Wigler et al., Proc. Natl. Acad. Sci. (U.S.A.) 77, 3567 (1980). |
Continuations (2)
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Number |
Date |
Country |
Parent |
149990 |
Jan 1988 |
|
Parent |
459153 |
Jan 1983 |
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