Primer sets for analyzing cytochrome P450 isoenzymes expression

• US 6,127,117 A
• Filed: 05/13/1997
• Issued: 10/03/2000
• Est. Priority Date: 05/13/1996
• Status: Expired due to Fees

• Current Assignee: GD Searle LLC (Pfizer Inc.)
• Original Assignee: GD Searle LLC (Pfizer Inc.)
• Inventors: Morris, Dale Lynn, Davila, Julio Cesar
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/855,583
• Time in Patent Office: 1,239 Days
• Field of Search: 536/22.1, 536/24.33, 435/91.2, 435/91.51, 435/6
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/142 Toxicological screening, e.g. expression profiles which identify toxicity
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Hepatocyte culturing system, primer sets and an analytical method for selectively detecting and quantitatively assessing the levels of mRNA expression of the major isoenzymes of cytochrome P450 (CYP450 1A1, 1A2, 2B1/2, 2C11, 2E1, 3A1, 3A2 and 4A1), fatty acyl-CoA oxidase (FACO) and select Phase II conjugating enzymes (UDPGT, GST and ST) in the rat using specific 5'"'"' and 3'"'"' oligonucleotide primers and reverse transcriptase-polymerase chain reaction. The method closely reproduces the expression obtained from rat liver tissue following treatment with the same enzyme inducers. Constitutive and inducible expression was maintained by resuspending, culturing and then overlaying adult rat hepatocytes with an extracellular matrix such as Matrigel®.

Claims

1. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 1A1 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of CTGGTTCTGGATACCCAGCTG (SEQ ID NO;

1) and CCTAGGGTTGGTTACCAGG (SEQ ID NO;

2).

10. A method for specifically detecting expression of DNA encoding a target enzyme selected from the group consisting of cytochrome P450 isoenzymes 1A1, 1A2, 2B1, 2B1/2, 2C11, 2E1, 3A1, 3A2, or 4A1 in a tissue following exposure to drugs or chemicals, the method comprising the steps of:

a) providing a sample of RNA extracted from the tissue;

b) converting the sample of RNA to cDNA using reverse transcriptase and oligo d(T)_15-18, primers;

c) amplifying the cDNA using polymerase chain reaction and a primer set of any one of claims 1-8 and 9 to provide amplified cDNA; and

d) separating and detecting the amplified cDNA to detect expression of said target enzyme.

11. The method of claim 10, wherein said RNA is extracted from rat liver.

2. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 1A2 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of GTCACCTCAGGGAATGCTGTG (SEQ ID NO;

3) and GTTGACAATCTTCTCCTGAGG (SEQ ID NO;

4).

3. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 2B1/2 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of GAGTTCTTCTCTGGGTTCCTG (SEQ ID NO;

5) and ACTGTGGGTCATGGAGAGCTG (SEQ ID NO;

6).

4. A primer set for specifically detecting expression of DNA of cytochrome P450 isoenzyme 2C11 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of CTGCTGCTGCTGAAACACGTG (SEQ ID NO;

     7) and GGATGACAGCGATACTATCAC (SEQ ID NO;

8).

5. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 2E1 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of CTCCTCGTCATATCCATCTG (SEQ ID NO;

9) and GCAGCCAATCAGAAATGTGG (SEQ ID NO;

10).

6. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 3A1 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of ATCCGATATGGAGATCAC (SEQ ID NO;

11) and GAAGAAGTCCTTGTCTGC (SEQ ID NO;

12).

7. A primer set for specifically detecting expression of DNA encoding cytochrome P450 isoenzyme 3A2 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of CGACTTGGAACCCATAGAC (SEQ ID NO;

13) and GGCTTAGGGAGATTTGACATG (SEQ ID NO;

14).

8. A primer set for specifically detecting expression of DNA of cytochrome P450 isoenzyme 4A1 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of GGTGACAAAGAACTACAGC (SEQ ID NO;

15) and AGAGGAGTCTTGACCTGCCAG (SEQ ID NO;

16).

9. A primer set for specifically detecting expression of DNA of cytochrome P450 isoenzyme 2B1 in a tissue following exposure to drugs or chemicals, the primer set comprising:

oligonucleotides which consist essentially of CAACCCTTGATGACCGCAGT (SEQ ID NO;

23) and GGAAGTGTTCAGGATTGAAGC (SEQ ID NO;

24).