C/EBPalpha gene targeting constructs and uses thereof

• US 20050100924A1
• Filed: 11/06/2003
• Published: 05/12/2005
• Est. Priority Date: 11/06/2003
• Status: Abandoned Application

• Current Assignee: National University of Singapore
• Original Assignee: National University of Singapore
• Inventors: Wang, Nai-dy
• Application Number:
  US10/703,224
• Publication Number:
  US 20050100924A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators

Abstract

A DNA construct for the expression of a functional mammalian C/EBPαcomprising the following components: (i) a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus (ii) a DNA molecule encoding the C/EBPα; and (iii) a second region having homology with a region within the gene locus. The DNA construct is used for the preparation of non-human knock-in animals.

Claims

1. A DNA construct comprising:

a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus;

a DNA molecule encoding C/EBPα

, the DNA molecule located 3′

of the first region; and

a second region having homology with a region within the gene locus, the second region located 3′

of the DNA molecule;

wherein the construct is operable to express a functional C/EBPα

protein.

2. The construct of claim 1, wherein the DNA molecule encodes human C/EBPα

.

3. The construct of claim 1, wherein the DNA molecule encodes mouse C/EBPα

.

4. The construct of claim 1, wherein the tumor-specific promoter gene comprises the AFP promoter gene and the gene locus comprises the AFP gene locus.

5. The construct of claim 1, further comprising a selectable marker, and identification tag, or a combination thereof.

6. The construct of claim 1, wherein the construct is inserted in an expression vector.

7. The construct of claim 6, wherein the expression vector further comprises:

a DNA molecule having the sequence of SEQ ID NO;

1; and

a DNA molecule having the sequence of SEQ ID NO;

2.

8. The construct of claim 1, wherein the construct is located within a host cell.

9. The construct of claim 8, wherein the host cell is present in a cell culture.

10. The construct of claim 8, wherein the host cell is an animal hepatocyte or pneumocyte.

11. The construct of claim 10, wherein the host cell is a human cell.

12. The construct of claim 8, wherein the host cell is a tumor-resistant cell.

13. The construct of claim 1, wherein the construct is present in a pharmaceutical preparation.

14. The construct of claim 13, wherein the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, a diluent, or a combination thereof.

15. The construct of claim 10, wherein the host cell is a hepatocyte suitable for grafting in an area in need of transplant.

16. The construct of claim 1, wherein the construct is present in a hepatocyte bioreactor.

17. A process for introducing a construct into an area in need of the construct comprising:

preparing a DNA construct operable to express functional mammalian C/EBPα

protein, the construct including;

a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus;

a DNA molecule encoding C/EBPα

, the DNA molecule located 3′

of the first region; and

a second region having homology with a region within the gene locus, the second region located 3′

of the DNA molecule; and

administering the construct to an area in need of the construct.

18. The method of claim 17, wherein the further comprising:

modifying hepatocytes to include the construct, wherein the modifying renders the hepatocytes tumor-resistant; and

administering the hepatocyes to an area in need of transplant.

19. The method of claim 18, wherein administering the hepatocytes further comprises supplying an autograft.

20. The method of claim 18, wherein administering the hepatocytes further comprises supplying an allograft.

21. The method of claim 18, further comprising preparing hepatocyte aggregates.

22. The method of claim 18, further comprising preparing hepatocyte grafts.

23. The method of claim 17, wherein the area in need of the construct is located within an animal.

24. The method of claim 23, wherein the animal was born prematurely and suffers from liver glycogen-insufficiency.

25. The method of claim 24, wherein the animal is a human.

26. The method of claim 23, further comprising:

injecting the construct into the animal; and

treating liver glycogen insufficiency in the animal.

27. The method of claim 23, wherein the animal comprises a cancer, and wherein the cancer produces elevated levels of AFP.

28. The method of claim 27, wherein the animal is a human.

29. The method of claim 23, further comprising:

injecting the construct into the animal; and

treating a cancer in the animal, wherein the cancer produces elevated levels of AFP.

30. The method of claim 23, wherein the animal exhibits one or more conditions associated with underdeveloped lung tissue.

31. The method of claim 30, wherein the animal is a human.

32. The method of claim 23, further comprising:

injecting the construct into the animal; and

treating one or more symptoms of underdeveloped lung tissue in the animal.

33. The method of claim 32, further comprising accelerating maturation of underdeveloped lung tissues.

34. A non-human knock-in animal comprising a DNA construct including:

a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus;

a DNA molecule encoding C/EBPα

, the DNA molecule located 3′

of the first region; and

a second region having homology with a region within the gene locus, the second region located 3′

of the DNA molecule;

wherein the construct is operable to express a functional C/EBPα

protein.

35. The animal of claim 34, wherein the animal is a mouse.

36. The animal of claim 34, wherein the construct is transcribed in the fetal liver at an earlier point than normal.

37. The animal of claim 34, wherein the construct expresses C/EBPα

mRNA prior to endogenous C/EBPα

production.

38. A tumor resistant cell comprising a DNA construct including:

a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus;

a DNA molecule encoding C/EBPα

, the DNA molecule located 3′

of the first region; and

a second region having homology with a region within the gene locus, the second region located 3′

of the DNA molecule;

wherein the construct is operable to express a functional C/EBPα

protein; and

wherein the tumor resistant cell is isolated from a knock-in animal including the DNA construct.

39. The tumor resistant cell of claim 38, wherein the cell is a hepatocyte or a pneumocyte.

40. The tumor resistant cell of claim 38, wherein the cell is a pluripotent, embryonic, or adult animal stem cell.

41. The tumor resistant cell of claim 38, wherein the cell is a human cell.

42. The tumor resistant cell of claim 38, wherein the cell is derived from a stem cell.

43. A method for in vivo tumor screening comprising:

modifying a non-human animal to express a DNA construct including;

a first region having homology with an endogenous tumor-specific promoter gene sequence of a gene locus;

a DNA molecule encoding C/EBPα

, the DNA molecule located 3′

of the first region; and

a second region having homology with a region within the gene locus, the second region located 3′

of the DNA molecule;

wherein the construct is operable to express a functional C/EBPα

protein;

administering to the animal one or more carcinogens; and

identifying one or more tumors, wherein the tumors are inhibited or suppressed by the function of C/EBPα

protein.

44. The method of claim 43, further comprising modulating growth of one or more tumors.