Claims
- 1. A human cell composition comprising PKR-overexpressing cells, prepared by the process of:
(a) providing a parental cell line capable of expressing PKR and one or more cytokines; (b) subjecting said parental cell line to limiting dilution cloning in a manner effective to generate a plurality of subclones; (c) assaying said plurality of subclones for PKR expression; and (d) selecting a PKR overexpressing subclone identified by a PKR expression level which is at least 2-fold (2×) greater than the PKR expression level of said parental cell line and growing said subclone to generate a PKR overexpressing cell line.
- 2. The cell composition of claim 1, wherein the preparative process further comprising:
(e) priming said PKR overexpressing cells; and (f) growing said primed PKR overexpressing cells in a manner effective to generate a PKR overexpressing cell line characterized by enhanced expression of one or more cytokines.
- 3. The cell composition of claim 2, wherein said preparative process further comprising treating said primed, PKR overexpressing cells before said growing (f).
- 4. The cell composition of claim 1, wherein said parental cell line is subjected to limiting dilution cloning at least 3 times.
- 5. The cell composition of claim 2, wherein said priming includes culturing the cells with phorbal myristate acetate (PMA) or sodium butyrate.
- 6. The cell composition of claim 3, wherein said treating includes non-viral induction with dsRNA.
- 7. The cell composition of claim 3, wherein said treating includes viral induction with Sendai virus.
- 8. The cell composition of claim 3, wherein said one or more cytokines are selected from the group consisting of α-interferon, β-interferon, TNF-β, IL-6 and IL-8, IL-10, IFN-gamma, IL-12 and GM-CSF.
- 9. The cell composition of claim 3, wherein said one or more cytokines include α- or β-interferon.
- 10. A human cell composition for use in cytokine production, comprising:
PKR overexpressing cells which have been treated and induced in a manner effective to result in enhanced PKR and cytokine production, wherein the cells are characterized by a level of expression or production of one or more cytokines that is at least 2-fold greater than that produced by the corresponding parental cell line.
- 11. A method for producing a cell line that overexpresses PKR, said method comprising:
(a) providing a parental cell line capable of expressing PKR and one or more cytokines; (b) subjecting said parental cell line to limiting dilution cloning in a manner effective to generate a plurality of subclones; (c) assaying said plurality of subclones for PKR expression; and (d) selecting a PKR overexpressing subclone identified by a PKR expression level which is at least 2-fold (2×) greater than the PKR expression level of said parental cell line; and (e) growing said subclone to generate a PKR overexpressing cell line.
- 12. The method according to claim 11 further comprising:
priming said PKR overexpressing cell line before said growing;
- 13. The method according to claim 12 further comprising:
inducing said primed, PKR overexpressing cell line before said growing.
- 14. The method of claim 12, wherein said priming includes culturing said PKR overexpressing cells with phorbal myristate acetate (PMA) or sodium butyrate.
- 15. The method of claim 13, wherein said inducing includes non-viral induction with dsRNA and viral induction with Sendai virus.
- 16. The method of claim 11, wherein said growing is in a manner effective to generate a PKR overexpressing cell line characterized by enhanced expression of one or more cytokines.
- 17. The method according to claim 16, wherein said one or more cytokines are selected from the group consisting of α-interferon, β-interferon, TNF-β, IL-6 and IL-8.
- 18. The method according to claim 16, wherein said one or more cytokines include α- or β-interferon.
- 19. A human cell composition for enhanced cytokines production comprising PKR-overexpressing cells, prepared by a process comprising:
(a) providing a parental cell line capable of expressing PKR and a heterologous nucleic acid construct that comprises a first coding sequence for a biologically active form of human PKR and a second coding sequence for a selectable marker; (b) introducing said heterologous nucleic acid sequence construct into said parental cell line in manner effective to result in expression of PKR and said selectable marker; (c) culturing said transformed parental cells under conditions effective to enhance expression of PKR; (d) selecting transformed, PKR overexpressing cells, wherein said PKR overexpressing cells are identified by a PKR expression level which is at least 2-fold (2×) greater than the PKR expression level of the non-transformed parental cell line; (e) growing said selected PKR overexpressing cells; and (f) repeating said selection and growing to generate PKR overexpressing progeny cell lines characterized by enhanced expression of one or more cytokines.
- 20. The cell composition of claim 19, wherein said preparative process further comprising:
priming said PKR overexpressing cells and treating said primed, PKR overexpressing cells before said growing.
- 21. The cell composition of claim 20, wherein said priming includes culturing the cells with phorbal myristte acetate (PMA) or sodium butyrate.
- 22. The cell composition of claim 20, wherein said treating includes non-viral induction with dsRNA or viral induction with Sendai virus.
- 23. The cell composition of claim 19, wherein said one or more cytokines are selected from the group consisting of α-interferon, β-interferon, TNF-β, IL-6 and IL-8, IL-10, IFN-gamma, IL-12 and GM-CSF.
- 24. The cell composition of claim 19, wherein said one or more cytokines include α- or β-interferon.
Parent Case Info
[0001] This application claims priority of U.S. Provisional Patent Application Serial No. 60/256,586 filed Dec. 19, 2000, which is incorporated in its entirety herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60256586 |
Dec 2000 |
US |