Immortalized cell lines and methods of making the same

• US 6,458,593 B1
• Filed: 01/21/2000
• Issued: 10/01/2002
• Est. Priority Date: 01/21/1999
• Status: Expired due to Fees

• Current Assignee: Vitro Diagnostics, Inc.
• Original Assignee: Vitro Diagnostics, Inc.
• Inventors: Burnett, Bruce P., Gill, John Charles IV, Hedlund, Tammy E., Musick, James R.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/489,085
• Time in Patent Office: 984 Days
• Field of Search: 435/458, 435/467, 435/366
• US Class Current: 435/458
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0616 Pituitary gland

Abstract

The present invention relates to methods of immortalizing various primary cell cultures, including pituitary cells, neurons, beta islet cells, glial cells, corneal epithelial cells and follicular stellate cells. The primary cells are transfected with a vector containing an establishment oncogene, resulting in non-transformed immortalized cells. The primary cells and/or the subsequent immortalized cells are cultured in a defined media containing one or more environmental factor(s) that control the proliferation and/or differentiation of the cells.

Claims

1. A method of immortalizing non-transforrned cells comprising the steps of:

(a) obtaining a primary culture of cells;

(b) transfecting said primary cells with a vector containing a foreign gene to produce transfected, immortalized cells that are not transformed; and

(c) isolating said transfected cells from non-transfected cells to obtain a culture of immortalized cells, wherein said immortalized cells are pituitary cells or follicular stellate cells.

2. The method of claim 1, wherein said primary cells are transfected by cationic liposomes, retroviruses or immunoliposomes.

3. The method of claim 1, wherein said primary cells are transfected by immunoliposomes.

4. The method of claim 1, wherein said primary cells are hormone-producing cells.

5. The method of claim 1, wherein said primary cells are isolated with immobilized antibody specific for a hormone or peptide hormone receptor expressed by pituitary cells prior to transfection.

6. The method of claim 1, wherein said primary cells are cultured with an extracellular matrix protein.

7. The method of claim 6, wherein said exracellular matrix protein is fetuin, matrix, or disrupted MCF-7 cells.

8. The method of claim 6, wherein said extracellular matrix protein is fetuin.

9. The method of claim 1, wherein said primary cells are pituitary cells.

10. The method of claim 9, wherein said pituitary cells are lactotropes, somatotropes, thyrotropes, gonadotropes, corticotropes or melanotropes.

11. The method of claim 1, wherein said primary cells are human cells.

12. The method of claim 1, further comprising culturing said primary cells with at least one environmental factor to control proliferation or differentiation prior to or after transfection.

13. The method of claim 12, wherein said environmental factor is a growth factor or other factor controlling the expression of the foreign gene.

14. The method of claim 13, wherein said growth factor is activin, tri-iodothyronine, galinin, nerve growth factor, leukemia inhibitory factor, hepatocyte growth factor, acidic or basic fibroblast growth factor, platelet-derived growth factor, pituitary adenylate cyclase-activating polypeptide, vasopressin, retinoic acid, vasoactive intestinal polypeptide or gonadotropin releasing hormone (GnRH).

15. The method of claim 1, wherein said vector is a retroviral expression vector.

16. The method of claim 15, wherein said retroviral expression vector is pLNCX2 or pLXSN.

17. The method of claim 16, wherein said retroviral expression vector contains an inducible promoter.

18. The method of claim 17, wherein said inducible promoter is derived from beta galactosidase, T7, mouse metallothionein, glucocorticoid or tetracycline response elements.

19. The method of claim 1, wherein said foreign gene is an establishment oncogene.

20. The method of claim 19, wherein said establishment oncogene is a viral oncogene, a cellular proto-oncogene, a tumor suppressor gene or a derivative thereof.

21. The method of claim 20, wherein said viral oncogene is large T antigen of SV40, an oncogene from the Epstein-Barr virus, or E7 gene of human papilloma virus.

22. The method of claim 21, wherein said viral oncogene is large T antigen of SV40.

23. The method of claim 20, wherein said cellular proto-oncogene is gsp, gip2, myc, fos or pituitary tumor-transforming gene.

24. The method of claim 20, wherein said cellular proto-oncogene is myc.

25. The method of claim 1, further comprising transfecting said primary cells with an anti-apoptosis gene.

26. The method of claim 25, wherein said anti-apoptosis gene is a Bc12 gene.

27. The method of claim 1, wherein said immortalized cells remain viable at least at a 5-fold greater passage number than senescence of non-immortalized primary cells under similar culture conditions.

28. An immortalized cell line produced by the method of claim 1.

29. The immortalized cell line of claim 28, wherein said immortalized cell line is a follicular stellate cell line.

30. The immortalized cell line of claim 28, wherein said immortaliedcell line is a pituitarcell line.

31. The immortalized cell line of claim 28, wherein said immortalized cell line is cultured in media free of other cell lines.

32. The immortalized cell line of claim 30, wherein said immortalized cell line produces prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, adrenocorticotropic hormone or melanocyte-stimulating hormone.

33. The method of claim 24, wherein said myc gene is contained within a vector that comprises an inducible promoter.

34. The method of claim 33, wherein said inducible promoter is derived from beta galactosidase, T7, mouse metallothionein, glucocorticoid or tetracycline response elements.

35. The method of claim 1, wherein said vector comprises an inducible promoter.

36. The method of claim 35, wherein said inducible promoter is derived from beta galactosidase, T7, mouse metallothionein, glucocorticoid or tetracycline response elements.

37. The method of claim 1, wherein said vector comprises endogenous promoter sequences specific to normal pituitary cell expression.

38. The method of claim 37, wherein said promoter sequences are pituitary hormone promoter sequences or the thereof.