التفاصيل البيبلوغرافية
| العنوان: |
GENE REPORTER ASSAY, KIT AND CELLS WITH IMPROVED SENSITIVITY AND/OR SPECIFICITY FOR DETERMINING THE LEVEL OF AN EXTRACELLULAR SIGNAL |
| Document Number: |
20080138818 |
| تاريخ النشر: |
June 12, 2008 |
| Appl. No: |
11/928965 |
| Application Filed: |
October 30, 2007 |
| مستخلص: |
The present invention provides a reporter gene containing cell line with increased specificity and/or sensitivity for a particular extracellular signal of interest so that it can be used in a gene-reporter assay to accurately determine the presence and/or level of the extracellular signal of interest in the presence of other extracellular signals that are capable of activating the same signal transduction pathway as the extracellular signal of interest or that are capable of activating another signal transduction pathway capable of modulating the transcription of the reporter gene. |
| Inventors: |
TOVEY, Michael G. (Paris, FR); LALLEMAND, Christophe (Paris, FR) |
| Claim: |
1. In a cell line transformed with a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to a transcriptional control element that is activated as part of the signal transduction pathway initiated by a first cell surface molecule or complex in response to a first extracellular signal, which signal transduction pathway includes a transcription factor that binds to the transcriptional control element so as to activate said transcriptional control element and thereby regulate transcription of the reporter gene, the improvement whereby the sensitivity and/or the specificity of the response of the cell line to the extracellular signal is improved, wherein: a) said transcription control element is a modification of a naturally occurring transcriptional control element that is activated as part of the signal transduction pathway initiated by said first cell surface molecule or complex in response to said first extracellular signal, or is a synthetic promoter comprising an optimal number of response elements specific for said transcriptional factor activated by said first cell surface molecule or complex but lacking response elements for other transcription factors, such that the sensitivity and/or specificity of the transcriptional control element is improved relative to the naturally occurring transcriptional control element; and/or b) the cells of said cell line lack a second cell surface molecule that responds to, or is part of a complex that responds to, a second extracellular signal, which second extracellular signal, if said second cell surface molecule were present, would cause the initiation of a signal transduction pathway that modulates the transcription of said reporter gene. |
| Claim: |
2. The cell line of claim 1, wherein said modification of a naturally occurring transcriptional control element is obtained by site directed mutagenesis of said naturally occurring transcriptional control element and selection for a modification that improves the sensitivity and/or specificity of the naturally occurring transcriptional control element. |
| Claim: |
3. The cell line of claim 1, wherein said modification of a naturally occurring transcriptional control element comprises a synthetic nucleotide sequence that comprises a tandem repeat of the naturally occurring or consensus sequence of the binding site for said transcription factor while lacking binding sites for other transcription factors. |
| Claim: |
4. The cell line of claim 3, wherein said transcription factor is NFκB. |
| Claim: |
5. The cell line of claim 4, wherein said tandem repeat of the binding site for transcription factor NFκB consists of the sequence of SEQ ID NO:2. |
| Claim: |
6. The cell line of claim 1, wherein the cells of said cell line naturally exist without said second cell surface molecule. |
| Claim: |
7. The cell line of claim 6, wherein said first extracellular signal is tumor necrosis factor-α (TNFα) and said second extracellular signal is interferon-γ (IFNγ) and/or interleukin-2 (IL2). |
| Claim: |
8. The cell line of claim 1, wherein the cells of said cell line have been genetically engineered to knock out said second cell surface molecule. |
| Claim: |
9. The cell line of claim 8, wherein said first extracellular signal is interferon-γ (IFNγ) and said second extracellular signal is interferon-α (IFNα) and/or interferon-β (IFN β). |
| Claim: |
10. The cell line of claim 1, wherein at least the extracellular portion of said first cell surface molecule or complex is that of a first species cell surface molecule or complex and the cells of said cell line are cells of a second species that have been genetically engineered to knock in said first cell surface molecule or complex. |
| Claim: |
11. The cell line of claim 1, wherein said first and/or second cell surface molecule or complex is a cell surface receptor. |
| Claim: |
12. The cell line of claim 1, wherein said first and/or second cell surface molecule or complex is a pattern recognition receptor. |
| Claim: |
13. The cell line of claim 1 in a frozen state, wherein said cell line has the property that it will maintain signal transduction activity of said signal transduction pathway initiated by said first cell surface molecule or complex for at least one hour after being thawed but will lose said signal transduction activity and undergo cellular death in no more than about 30 days at a temperature above freezing after being thawed. |
| Claim: |
14. A kit for determining the presence and/or level in a sample of an extracellular signal that activates the signal transduction pathway of a cell surface molecule or complex, comprising: a testing device having a plurality of wells; and a reagent containing a plurality of cells of the cell line of claim 1. |
| Claim: |
15. The kit of claim 14, wherein said testing device is a microtiter plate. |
| Claim: |
16. The kit of claim 14, wherein said reagent is disposed in the wells of said testing device. |
| Claim: |
17. The kit of claim 14, wherein the extracellular signal whose level in the sample is to be determined is IFNγ. |
| Claim: |
18. The kit of claim 14, wherein the extracellular signal whose level in the sample is to be determined is TNFα. |
| Claim: |
19. A method for determining the level in a sample of an extracellular signal that activates the signal transduction pathway of a cell surface molecule or complex, comprising: incubating cells of the cell line of claim 1 with a sample in which the level of an extracellular signal that activates the signal transduction activity of a cell surface molecule is sought to be determined; and determining the level of expression of the reporter gene product in the cells to thereby determine the level in the sample of the extracellular signal that activates the signal transduction activity of the cell surface molecule or complex. |
| Claim: |
20. The method of claim 19, wherein the cell surface molecule is a Type II interferon receptor and the extracellular signal is IFNγ. |
| Claim: |
21. The method of claim 19, wherein the cell surface molecule is a TNFα receptor and the extracellular signal is TNFα. |
| Claim: |
22. The method of claim 19, wherein determining the level in a sample of the extracellular signal that activates the signal transduction activity of the cell surface molecule or complex indirectly determines the level of an antagonist to the extracellular signal of interest or the level of an antibody against the antagonist. |
| Claim: |
23. The method of claim 22, wherein the antagonist is a TNFα antagonist. |
| Current U.S. Class: |
435/6 |
| Current International Class: |
12; 12; 12 |
| رقم الانضمام: |
edspap.20080138818 |
| قاعدة البيانات: |
USPTO Patent Applications |