Stably-transformed cells expressing human thiopurine methyltransferase

• US 5,470,737 A
• Filed: 10/03/1994
• Issued: 11/28/1995
• Est. Priority Date: 06/09/1993
• Status: Expired due to Term

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Wieben, Eric D., Szumlanski, Carol L., Honchel, Ronald, Weinshilboum, Richard M., Wood, Thomas C., Otterness, Diane M., Aksoy, Ibrahim A.
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US08/317,707
• Time in Patent Office: 421 Days
• Field of Search: 536/23.1, 536/23.2-23.5, 435/240.21, 435/193, 435/172.3, 435/320.1, 435/240.2, 435/91.4, 435/91.2, 930/240, 935/9, 935/11, 935/14, 935/32, 935/40
• US Class Current: 435/371
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)

Abstract

A recombinant DNA sequence is provided which encodes human thiopurine methyl transferase (TPMT), as well as a mammalian cell line having said recombinant DNA sequence incorporated into the genome thereof, so that the cells express high levels of human TPMT activity.

Claims

1. A mammalian cell line, the genome of which has been augmented by chromosomally integrated non-native DNA encoding mammalian thiopurine methyltransferase, wherein said DNA hybridizes with DNA complementary to DNA having SEQ ID NO:

1 under the stringency conditions of hybridization in buffer containing 20% formamide, 5×

Denhardt'"'"'s, 6×

SSC, 100 mg/ml RNA and 0.05% sodium pyrophosphate at 42°

C., followed by washing at 60°

C. and 1×

SSC, 0.1% SDS.

2. The transgenic mammalian cell line of claim 1 wherein the mammalian cell line is a primate cell line.

3. The transgenic mammalian cell line of claims 1 or 2 wherein the mammalian TPMT is human TPMT.

4. A mammalian cell line, the genome of which has been augmented by chromosomally integrated non-native DNA encoding human thiopurine methyl transferase having an amino acid sequence which is SEQ ID NO:

2.

5. The mammalian cell line of claim 4, wherein the non-native DNA has the sequence consisting of SEQ ID NO:

1.

6. An isolated and purified DNA encoding human thiopurine methyl transferase that hybridizes to DNA complementary to DNA having SEQ ID NO:

1 under the stringency conditions of hybridization in buffer containing 20% formamide, 5×

Denhardt'"'"'s, 6×

SSC, 100 mg/ml RNA and 0.05% sodium pyrophosphate at 42°

C., followed by washing at 60°

C. and 1×

SSC, 0.1% SDS.

9. An isolated and purified DNA which functions to express human thiopurine methyltransferase when introduced into a mammalian cell, wherein said DNA is not native to said cell and comprises the DNA of claims 6, 7 or 8, operably linked to a promoter functional in said mammalian cell.

7. An isolated and purified DNA encoding only the human thiopurine methyl transferase having an amino acid sequence which is SEQ ID NO:

2.

8. The DNA of claim 7 which has SEQ ID NO:

1.