التفاصيل البيبلوغرافية
| العنوان: |
Vector for the inducible expression of gene sequences |
| Document Number: |
20070122880 |
| تاريخ النشر: |
May 31, 2007 |
| Appl. No: |
11/550045 |
| Application Filed: |
October 17, 2006 |
| مستخلص: |
The invention relates to a nucleic acid construct that comprises a human EF-1α promoter operatively associated with the tetracycline repressor (TetR). The invention further provides cell lines that have been stably transfected with such construct, as well as methods of using said construct, for the inducible expression of a transgene or for the inducible silencing of a endogenous sequence in mammalian cells. |
| Inventors: |
GUILLOUF, Christel (Paris, FR); Spinglard, Anne (Nanterre, FR) |
| Assignees: |
Institut Curie (Paris, FR) |
| Claim: |
1. A nucleic acid construct that comprises a tetracycline repressor (TetR) operatively associated with a EF-1α promoter, preferably a human EF-1α promoter. |
| Claim: |
2. The nucleic acid construct of claim 1, which comprises (a) a tetracycline repressor (TetR), operatively associated with a EF-1α promoter, and a transcription termination sequence; (b) at least one positive selection gene, operatively associated with a promoter; (c) at least one replication origin for replication in a prokaryotic cell, and optionally at least one replication origin for replication in a eukaryotic cell. |
| Claim: |
3. The nucleic acid construct of claim 2, that is a circle plasmid vector comprising, in the following order, a human EF-1α promoter an intronic sequence that are upstream a tetracycline repressor (TetR), the latter being followed by a transcription termination polyA sequence; a replication origin in a prokaryotic cell, a replication origin in a eukaryotic cell, a promoter that drives the expression of a blasticidin resistance gene as the positive selection gene, followed by a transcription termination polyA sequence. |
| Claim: |
4. The nucleic acid construct of claim 3, that has the sequence set forth in SEQ ID NO:1, the construct being called pcDNA6/TR-EF-1α. |
| Claim: |
5. A host cell that comprises the nucleic acid construct of claim 1. |
| Claim: |
6. The host cell of claim 5, which is a eukaryotic cell, preferably a mammal cell. |
| Claim: |
7. The host cell of claim 6, that is an adherent or non adherent cell line. |
| Claim: |
8. The host cell of claim 6, which is a NIH 3T3 cell. |
| Claim: |
9. A method for inducibly expressing a gene sequence of interest in a cell, which method comprises providing a host cell that stably comprises the nucleic acid construct of claim 1, and transfecting said cell with a nucleic acid construct carrying a gene sequence of interest that is operatively associated with a promoter under the control of a Tet operator, wherein addition of tetracycline or tetracycline analog induces expression of the gene sequence of interest in the cell. |
| Claim: |
10. The method of claim 9, wherein the cell is a mammalian cell line. |
| Claim: |
11. A method for inducibly silencing an endogenous sequence of interest in a cell, which method comprises providing a host cell that stably comprises the nucleic acid construct of claim 1, and transfecting said cell with a nucleic acid construct carrying DNA sequences leading to the synthesis of SiRNAs directed against said endogenous sequence of interest, wherein said DNA sequences are operatively associated with a promoter under the control of a Tet operator, wherein addition of tetracycline or tetracycline analog induces synthesis of said SiRNAs and silencing of said endogenous sequence of interest. |
| Claim: |
12. The method of claim 11, wherein the cell is a mammalian cell line. |
| Current U.S. Class: |
435069/100 |
| Current International Class: |
12; 07; 07 |
| رقم الانضمام: |
edspap.20070122880 |
| قاعدة البيانات: |
USPTO Patent Applications |