DNA controlling miR-140 expression, and screening method of drugs using said DNA

Patent metadata

Field	Value
Publication number	US-9557327-B2
Application number	US-201314388609-A
Country	US
Kind code	B2
Filing date	Apr 2, 2013
Priority date	Apr 3, 2012
Publication date	Jan 31, 2017
Grant date	Jan 31, 2017

Abstract

An object of the present invention is to provide DNA which regulates the expression of miR-140, a reporter vector which contains the DNA, cells and animals into which the reporter vector is introduced and a screening system for drugs which control the expression of miR-140 and is also to contribute in the development of new therapeutic agents for cartilage diseases such as osteoarthritis and intervertebral disk degeneration using the screening system. The DNA sequence according to the present invention is able to express any gene in a site where miR-140 is expressed and, in addition, it is also able to be utilized for screening a drug which regulates the expression of miR-140. Moreover, the drug that is selected by the screening system of the present invention is expected as a therapeutic agent for cartilage diseases accompanied by abnormality of cartilage.

First claim

The invention claimed is:

1. A vector comprising:
a first DNA sequence that includes at least one of:
the nucleotide sequence of SEQ ID No. 1 or 2; and

a reporter gene downstream of the first DNA sequence.

2. The vector according to claim 1 , wherein the reporter gene is LacZ, β-glucosidase, β-glucuronidase, alkaline phosphatase, chloramphenicol acetyl transferase (CAT), luciferase or fluorescent protein.

3. An isolated cell into which the vector of claim 1 is introduced.

4. The isolated cell according to claim 3 , wherein the cell is any cell selected from the group consisting of a 293T cell, ATDC5 cell, SW1353 cell, primarily cultured chondrocyte, mesenchymal stem cell and embryonic stem cell (ES cell).

5. A non-human animal into which the vector of claim 1 is introduced.

6. The non-human animal according to claim 5 , wherein the animal is a mouse or a rat.

7. A method for screening a drug which regulates the expression of miR-140, comprising
detecting reporter activity in the cell of claim 3 in the presence of a drug; and
comparing the detected reporter activity with a control reporter activity detected in the absence of a drug to determine if the drug increases or decreases the reporter activity.

8. A method for screening a drug which regulates the expression of miR-140, the method comprising:
(a) contacting a drug with the cell of claim 3 ;
(b) detecting a reporter activity in the cell; and
(c) selecting a drug which increases or decreases the reporter activity by comparing with a control detected in the absence of a drug.

9. A method for screening a drug which regulates the expression of miR-140, the method comprising:
introducing the vector of claim 1 into a cell;
contacting a drug with the cell;
detecting a reporter activity in the cell; and
selecting a drug that increases or decreases the reporter activity by comparing with a control detected in the absence of a drug.

10. The vector according to claim 1 , wherein the nucleotide sequence of SEQ ID No. 1 or 2 is repeated in the vector from two to five times.

11. The vector according to claim 1 , wherein the first DNA sequence further includes a nucleotide sequence having at least 60% sequence identity with the nucleotide sequence of SEQ ID No. 3 or 4.

12. The vector according to claim 1 , wherein the first DNA sequence further includes a nucleotide sequence having at least 60% sequence identity with the nucleotide sequence of SEQ ID No. 5 or 6.

13. The vector according to claim 1 , wherein the first DNA sequence further includes a nucleotide sequence having at least 60% sequence identity with any of the nucleotide sequences selected form the group consisting of SEQ ID Nos. 7-10, 40, and 41.

14. The vector according to claim 11 , wherein the nucleotide sequence of the first DNA sequence includes the nucleotide sequence of SEQ ID No. 1 or 2 and has at least 70% sequence identity with the nucleotide sequence of SEQ ID No. 3 or 4.

15. The vector according to claim 12 , wherein the nucleotide sequence of the first DNA sequence includes the nucleotide sequence of SEQ ID No. 1 or 2 and has at least 70% sequence identity with the nucleotide sequence of SEQ ID No. 5 or 6.

16. The vector according to claim 13 , wherein the nucleotide sequence of the first DNA sequence includes the nucleotide sequence of SEQ ID No. 1 or 2 and has at least 70% sequence identity with any of the nucleotide sequences selected form the group consisting of SEQ ID Nos. 7-10, 40, and 41.

17. A method of detecting reporter activity, comprising:
introducing the vector of claim 1 into a cell; and
detecting reporter activity in the cell.

Assignees

• Nat Center For Child Health And Dev
• Nippon Zoki Pharmaceutical Co

Inventors

• Asahara Hiroshi
• Yamashita Satoshi
• Naiki Mitsuru
• Abe Kentaro

Classifications

• A01K2227/105

  Murine · CPC title

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• A01K2217/05

  Animals comprising random inserted nucleic acids (transgenic) · CPC title

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title