Expression Systems Using Mammalian Beta-Actin Promoter

• US 20080250514A1
• Filed: 12/03/2004
• Published: 10/09/2008
• Est. Priority Date: 12/03/2003
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Tsunoda, Hiroyuki, Habu, Kiyoshi
• Granted Patent: US 8,298,816 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/48 regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Promoter activities were examined by comparing combinations of promoters and enhancers derived from various genes. A hybrid promoter comprising a combination of a CMV enhancer and a mammalian β-actin promoter, or the post-transcriptional regulatory region of the genomic sequence Woodchuck Hepatitis Virus (WPRE) and a mammalian β-actin promoter was found to be stronger than existing promoters. Furthermore, the activities of the β-actin promoters could be enhanced by coexpressing the oncogene product Ras, which is a transactivator.

Claims

1. A DNA construct, wherein a mammalian β

-actin promoter is operably linked to an enhancer.

2. The DNA construct of claim 1, wherein the enhancer is derived from Cytomegalovirus (CMV).

3. The DNA construct of claim 1, wherein the enhancer is Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

4. The DNA construct of claim 1, wherein the mammalian β

-actin promoter is a rodent β

-actin promoter.

5. The DNA construct of claim 2, wherein the CMV enhancer comprises the nucleotide sequence shown in SEQ ID NO:

4 and the mammalian β

-actin promoter comprises the nucleotide sequence shown in SEQ ID NO;

2.

6. The DNA construct of claim 3, wherein the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) comprises the nucleotide sequence shown in SEQ ID NO:

3 and the mammalian β

-actin promoter comprises the nucleotide sequence shown in SEQ ID NO;

2.

7. A vector comprising the DNA construct of claim 1.

8. The vector of claim 7, comprising a DNA having a desired DNA operably linked downstream of the mammalian β

-actin promoter.

9. The vector of claim 7, comprising and capable of expressing a DNA encoding a transactivator.

10. The vector of claim 9, wherein the transactivator is an oncogene product.

11. The vector of claim 10, wherein the oncogene product is Ras.

12. The vector of claim 8, wherein the desired DNA encodes a desired protein.

13. A cell comprising the vector of claim 8.

14. A cell comprising the vector of claim 8, wherein the oncogene is activated.

15. The cell of claim 14, into which the vector comprising the gene encoding the transactivator is introduced.

16. The cell of claim 14, which is a transformed cell.

17. The cell of claim 13, which is a mammalian cell.

18. The cell of claim 17, which is a rodent cell.

19. The cell of claim 13, which is derived from the same animal order as that from which the β

-actin promoter is derived.

20. The cell of claim 19, which is derived from the same animal species as that from which the β

-actin promoter is derived.

21. A non-human transgenic animal into which the vector according to claim 8 has been introduced.

22. A totipotent cell into which the vector of claim 8 is introduced.

23. A method for producing a desired protein, which comprises culturing a cell comprising the vector of claim 12;

and harvesting the expressed protein from the cultured cell or medium.

24. The method of claim 23, which comprises adding a transactivator to the medium.

25. A method for expressing a desired DNA in a host cell, which comprises introducing the vector of claim 8 into the host cell derived from the same animal order as that from which the β

-actin promoter in the vector is derived.

26. The method of claim 25, wherein the host cell is derived from the same animal species as that from which the β

-actin promoter in the vector is derived.

27. A method for expressing a desired DNA in a host cell, which comprises introducing the vector of claim 8 and a vector comprising and capable of expressing a DNA encoding a transactivator into a host cell which is derived from the same species as that from which the β

-actin promoter in the vector of claim 8 is derived.

28. The method of claim 25, wherein the host cell is a mammalian cell.

29. The method of claim 25, wherein the host cell is a rodent cell.

38. The method of claim 27, wherein the host cell is a mammalian cell.

39. The method of claim 27, wherein the host cell is a rodent cell.

30. A method for increasing the expression level of a desired DNA in a host cell, which comprises inserting upstream of the desired DNA a β

-actin promoter derived from the same animal order as that from which the host cell is derived.

31. The method of claim 30, wherein the β

-actin promoter is derived from the same animal species as that from which the host cell is derived.

32. The method of claim 30, which further comprises inserting an enhancer.

33. The method of claim 32, wherein the enhancer is Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).

34. The method of claim 32, wherein the enhancer is a CMV enhancer.

35. The method of claim 30, which comprises inserting a gene encoding a transactivator gene.

36. The method of claim 30, wherein the host cell is a mammalian cell.

37. The method of claim 30, wherein the host cell is a rodent cell.