Gene reporter assay, kit, and cells for determining the presence and/or the level of a molecule that activates signal transduction activity of a cell surface protein
View Patent ↗The present invention relates to a commercializable cell and to a gene reporter assay method and a kit which use this cell to determine the presence and/or the level of a molecule that activates signal transduction activity of a cell surface protein. This cell is treated in such a manner that it will have a sufficiently long shelf life for its intended purpose, whereupon at the end of its useful shelf life or at the end of its use, i.e., in an assay, the cell undergoes cellular death.
1. A mammalian cell line transformed with a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to one or more transcriptional control elements that are regulated by the signal transduction activity of a cell surface Type I or Type II interferon receptor in response to an extracellular signal provided by a Type I and/or Type II interferon, said cell line having been treated with an antimitotic and pro-apoptotic agent so as to acquire the property that it will maintain said signal transduction activity for at least about 1 hour but will lose said signal transduction activity and undergo cellular death in no more than about 30 days at a temperature above freezing, wherein said anti-mitotic and pro-apoptotic agent is selected from the group consisting of vinblastine, 5-fluorouracil and γ-irradiation.
2. The mammalian cell line of claim 1 in a frozen state, wherein said cell line has been treated so as to acquire the property that it will maintain said signal transduction activity for at least about 1 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.
3. The mammalian cell line of claim 1 , wherein, substantially immediately after having been treated, said treated cell line has been resuspended in a solution containing a cryopreservative, and then frozen at about −80 °C.
4. The mammalian cell of claim 3 , wherein said cryopreservative is dimethylsulfoxide (DMSO) and said solution contains 10% DMSO.
5. The mammalian cell line of claim 3 , wherein said cryopreservative is a combination of 2.5% dimethylsulfoxide (DMSO) and 10% glycerol.
6. The mammalian cell line of claim 1 , wherein said anti-mitotic and pro-apoptotic agent is vinblastine.
7. The mammalian cell line of claim 1 , wherein said anti-mitotic and pro-apoptotic agent is 5-fluorouracil.
8. The mammalian cell line of claim 1 , wherein said anti-mitotic and pro-apoptotic agent is γ-irradiation and wherein said cell line has been irradiated with γ radiation at an intensity and for a sufficient amount of time such that said cell line maintains said signal transduction activity for at least about 1 hour but loses said signal transduction activity and undergoes cellular death in no more than about 30 days at a temperature above freezing.
9. The mammalian cell line of claim 8 , wherein said temperature above freezing is room temperature.
10. The mammalian cell line of claim 8 , wherein said intensity and amount of time of γ radiation is 6 to 12 (Gy).
11. The mammalian cell line of claim 1 , wherein said cell surface interferon protein is a Type I interferon receptor and said extracellular signal is provided by a Type I interferon.
12. The mammalian cell line of claim 11 , wherein said one or more transcriptional control elements comprise an interferon stimulatory response element (ISRE).
13. The mammalian cell line of claim 12 , wherein said ISRE comprises the nucleotide sequence of SEQ ID NO:5.
14. The mammalian cell line of claim 1 , wherein said cell surface interferon receptor is a Type II interferon receptor and said extracellular signal is provided by a Type II interferon.
15. The mammalian cell line of claim 14 , wherein said one or more transcriptional control elements comprise a gamma activated sequence (GAS).
16. The mammalian cell line of claim 1 , wherein said one or more transcriptional control elements comprise an interferon stimulatory response element (ISRE) and a gamma activated sequence (GAS).
17. The mammalian cell line of claim 1 , wherein said reporter gene product is selected from the group consisting of firefly luciferase, bacterial luciferase, jellyfish aequorin, enhanced green fluorescent protein (EGFP), chloramphenicol acetyltransferase (CAT), dsRED, β-galactosidase, and alkaline phosphatase.
18. The mammalian cell line of claim 1 , wherein said reporter gene product is firefly luciferase.
19. The mammalian cell line of claim 1 , wherein said reporter gene product is enhanced green fluorescent protein (EGFP).
20. The mammalian cell line of claim 1 , wherein said reporter gene product is jellyfish aequorin.
21. The mammalian cell line of claim 1 which is a human cell line.
22. The mammalian cell line of claim 21 which is a human promonocytic cell.
23. The mammalian cell line of claim 22 which is a PIL5 cell line.
24. A kit for determining the level in a sample of a Type I and/or Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor, comprising:
a testing device having a plurality of wells; and
a reagent containing a plurality of the cell of claim 1 .
25. The kit of claim 24 , wherein said testing device is a microtiter plate.
26. The kit of claim 24 , wherein said reagent is disposed in the wells of said testing device.
27. The kit of claim 24 , further comprising a set of instructions for using the kit to determine the level in a sample of a Type I and/or Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor.
28. A frozen mammalian cell transformed with a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to one or more transcriptional control elements that is regulated by the signal transduction activity of a cell surface Type I and/or Type II interferon receptor in response to an extracellular signal provided by a Type I and/or Type II interferon, said cell having been treated with an antti-mitotic and pro-apoptotic agent substantially immediately prior to freezing such that it will maintain said cell signal transduction activity for at least about 1 hour but but will lose said signal transduction activity and undergo cellular death in no more than about 30 days at a temperature above freezing, wherein said anti-mitptic and pro-apoptotic agent is selected from the group consisting of vinblastine, 5-fluorouracil and γ-irradiation.
29. A method for preparing the mammalian cell line of claim 1 , comprising:
transforming a mammalian cell line with a reporter gene construct comprising a nucleotide sequence encoding a reporter gene product operatively linked to one or more transcriptional control elements that is regulated by the signal transduction activity of a cell surface Type I and/or Type II interferon receptor in response to an extracellular signal provided by a Type I and/or Type II interferon; and
treating said transformed cell with an anti-mitotic and pro-apoptotic agent such that it will maintain said signal transduction activity for at least about 1 hour but will lose said signal transduction activity and undergo cellular death in no more than about 30 days at a temperature above freezing, wherein said anti-mitotic and pro-apoptotic agent is selected from the group consisting of vinblastine, 5-fluorouracil and γ-irradiation.
30. The method of claim 29 , wherein the mammalian cell line is a human cell line.
31. The method of claim 30 , wherein the human cell line is a human promonocytic cell line.
32. The method of claim 29 , wherein the cell surface Type I and/or Typre II interferon receptor is a Type I interferon receptor and the extracellular signal is Type I interferon.
33. The method of claim 29 , wherein the reporter gene product is selected from the group consisting of firefly luciferase, bacterial luciferase, jellyfish aequorin, enhanced green fluorescent protein (EGFP), chloramphenicol acetyltransferase (CAT), dsRED, β-galactosidase, and alkaline phosphatase.
34. The method of claim 29 , further including the step of freezing the treated cell line at a temperature and under conditions such that it will resume signal transduction after thawing.
35. The method of claim 29 , wherein said treating step comprises irradiating the transformed cell with γ-radiation at an intensity and for a sufficient time such that the irradiated cell line maintains the signal transduction activity of the cell surface Type I and/or Type II interferon receptor for a period of at least about 7 days but will lose said signal transduction activity and undergo cellular death in no more than 30 days at a temperature above freezing following irradiation.
36. The method of claim 35 , wherein, in said irradiating step, the transformed cell line is irradiated with about 6 to 12 Grays (Gy) of γ radiation.
37. The method of claim 35 , wherein the temperature above freezing is room temperature.
38. A method for determining the presence and/or the level in a sample of a Type I and/or Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor, comprising:
incubating the mammalian cell line of claim 1 , within the period of time that said cell line maintains said signal transuction activity, with a sample in which the presence and/or the level of a Type I and/or Type II interfeon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor and serves as the extracellular signal is sought to be determined; and
determining the level of expression of the reporter gene product to thereby determine the presence and/or the level in a sample of the Type I and/or Type II interferon that activates the signal transduction activity of the cell surface Type I and/or Type II interferon receptor.
39. The method of claim 38 , wherein the cell surface Type I and Type II interferon receptor is a Type I interferon receptor and the extracellular signal is a Type I interferon.
40. The method of claim 29 , further comprising the step of freezing the cell line at a temperature and under conditions such that it will resume signal transduction after thawing.
41. The method of claim 40 , wherein said step of freezing is carried out at a temperature in a range of about −20° C. to about −200° C. and the cells are subsequently stored at a temperature of about −20° C.
42. The method of claim 40 , wherein said step of freezing is carried out at a temperature of about −80° C.
43. The method of claim 40 , further comprising resuspending the treated cell in a solution containing a cryopreservative before said step of freezing.
44. The method of claim 43 , wherein the cryopreservative is a combination of DMSO and glycerol and the solution contains about 2.5% DMSO and 10% glycerol.
45. The method of claim 43 , wherein the solution comprises RPMI medium and 40% fetal bovine serum.
46. The method of claim 29 , wherein the anti-mitotic and pro-apoptotic agent is vinblastine.
47. The method of claim 29 , wherein the anti-mitotic and pro-apoptotic agent is 5-fluorouracil.
48. A method for determining the presence and/or the level in a sample of a Type I and/or Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor, comprising:
thawing a frozen cell in accordance with claim 2 ;
within the period of time that said thawed cell line maintains said signal transduction activity, incubating the thawed cell with a sample in which the presence and/or the level of a Type I and/or Type II interferon that activates the signal transduction activity of the cell surface Type I and/or Type II interferon and serves as the extracellular signal is sought to be determined; and
determining the level of expression of the reporter gene product to thereby determine the presence and/or the level in a sample of the Type I and/or Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor.
49. The method of claim 48 , wherein the cell surface Type I and/or Type II interferon receptor is a Type I interferon receptor and the extracellular signal is a Type I interferon.
50. The mammalian cell line of claim 1 , wherein said property is that it will maintain said signal transduction activity for at least one hour but will lose said signal transduction activity and undergo cellular death in no more than about 24 hours at a temperature above freezing.
51. The mammalian cell line of claim 1 , wherein said property is that is will maintain said signal transduction activity for at least one hour but will lose said signal transduction activity and undergo cellular death in no more than 14 days.
52. A composition comprising the cell line of claim 1 and a cryopreservative.
53. The composition of claim 52 , wherein said cryopreservative is dimethylsulfoxide (DMSO) and said solution contains 10% DMSO.
54. The composition of claim 52 , wherein said cryopreservative is a combination of 2.5% dimethylsulfoxide (DMSO) and 10% glycerol.
55. A kit for determining the level in a sample of a Type I and/ot Type II interferon that activates the signal transduction activity of a cell surface Type I and/or Type II interferon receptor, comprising:
a testing device having a plurality of wells; and
a reagent containing the mammalian cell line of claim 2 .