Method for assessing embryotoxicity

Patent metadata

Field	Value
Publication number	US-9783852-B2
Application number	US-99564209-A
Country	US
Kind code	B2
Filing date	Jun 2, 2009
Priority date	Jun 3, 2008
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

The present invention provides a method for assessing embryotoxicity of a chemical comprising: (1) a first step of measuring the expression level of one or more genes selected from among genes each comprising any of the nucleotide sequences of SEQ ID NOs: 1 to 78 and 101 to 230 and orthologous genes thereof in a sample from a non-human mammal or mammalian cell which has come into contact with a test chemical; and (2) a second step of comparing the measured value of the expression level of the gene in the sample obtained in the first step with a control value of the expression level of the gene and based on the difference assessing the level of the embryotoxicity of the test chemical in the sample; and so on.

First claim

The invention claimed is:

1. An in vitro method for assessing embryotoxicity of a chemical during myocardial cell differentiation comprising:
(A) culturing in vitro a first transformed mammalian embryonic stem cell in a medium for myocardial differentiation containing a test chemical, wherein said first transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Hand1—or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a first cultured cell;
(B) culturing in vitro a second transformed mammalian embryonic stem cell in a medium for myocardial differentiation not containing the test chemical, wherein said second transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Hand1—or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a second cultured cell;
(C) measuring the expression level of the reporter protein in the first cultured cell to obtain a measured value;
(D) measuring the expression level of the reporter protein in the second cultured cell to obtain a control value; and
(E) comparing the measured value with the control value obtained from the cultured cells on the day when the expression level of the reporter protein in the second cultured cell is the highest during myocardial cell differentiation, wherein when the measured value is lower than the control value, the test chemical is assessed to have embryotoxicity during myocardial cell differentiation.

2. The method according to claim 1 , wherein the medium used in the culturing of (A) contains the test chemical at a concentration not showing inhibition of cell proliferation.

3. The method according to claim 1 , wherein the measured value and the control value are obtained from the cultured cells on day 6 of the culturing.

4. The method according to claim 1 , wherein the reporter protein coding sequence is a luciferase coding sequence or a fluorescent protein coding sequence, and the promoter sequence is 5, 2 or 1 kb of the promoter region of the Hand1—or orthologous gene.

5. The method according to claim 1 , wherein the mammalian embryonic stem cell is from a mouse.

6. The method according to claim 1 , wherein the mammalian embryonic stem cell is seeded in a nonadherent U-bottom 96-well plate.

7. The method according to claim 6 , wherein the mammalian embryonic stem cell is seeded in a cell suspension of 15,000 cells/mL.

8. An in vitro method for assessing embryotoxicity of a chemical during myocardial cell differentiation comprising:
(A) culturing in vitro a first transformed mammalian embryonic stem cell in a medium for myocardial differentiation containing a test chemical, wherein said first transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Cmya1—or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a first cultured cell;
(B) culturing in vitro a second transformed mammalian embryonic stem cell in a medium for myocardial differentiation not containing the test chemical, wherein said second transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Cmya1—or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a second cultured cell;
(C) measuring the expression level of the reporter protein in the first cultured cell to obtain a measured value;
(D) measuring the expression level of the reporter protein in the second cultured cell to obtain a control value; and
(E) comparing the measured value with the control value obtained from the cultured cells on the day when the expression level of the reporter protein in the second cultured cell is the highest during myocardial cell differentiation, wherein when the measured value is lower than the control value, the test chemical is assessed to have embryotoxicity during myocardial cell differentiation.

9. The method according to claim 8 , wherein the medium used in the culturing of (A) contains the test chemical at a concentration not showing inhibition of cell proliferation.

10. The method according to claim 8 , wherein the measured value and the control value are obtained from the cultured cells on day 8 of the culturing.

11. The method according to claim 8 , wherein the reporter protein coding sequence is a luciferase coding sequence or a fluorescent protein coding sequence, and the promoter sequence is 5, 2 or 1 kb of the promoter region of the Cmya1—or orthologous gene.

12. The method according to claim 8 , wherein the mammalian embryonic stem cell is from a mouse.

13. An in vitro method for assessing embryotoxicity of a chemical during neural cell differentiation comprising:
(A) culturing in vitro a first transformed mammalian embryonic stem cell in a medium for neural differentiation containing a test chemical, wherein said first transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Reln—or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a first cultured cell;
(B) culturing in vitro a second transformed mammalian embryonic stem cell in a medium for neural differentiation not containing the test chemical, wherein said second transformed mammalian embryonic stem cell is a mammalian embryonic stem cell transformed with a reporter construct which comprises a Reln- or orthologous gene promoter sequence operatively linked to a heterologous reporter protein coding sequence, to obtain a second cultured cell;
(C) measuring the expression level of the reporter protein in the first cultured cell to obtain a measured value;
(D) measuring the expression level of the reporter protein in the second cultured cell to obtain a control value; and
(E) comparing the measured value with the control value obtained from the cultured cells on the day when the expression level of the reporter protein in the second cultured cell is the highest during neural cell differentiation, wherein when the measured value is lower than the control value, the test chemical is assessed to have embryotoxicity during neural cell differentiation.

14. The method according to claim 13 , wherein the medium used in the culturing of (A) contains the test chemical at a concentration not showing inhibition of cell proliferation.

15. The method according to claim 13 , wherein the measured value and the control value are obtained from the cultured cells on day 9 of the culturing.

16. The method according to claim 13 , wherein the reporter protein coding sequence is a luciferase coding sequence or a fluorescent protein coding sequence, and the promoter sequence is 5, 2 or 1 kb of the promoter region of the Reln- or orthologous gene.

17. The method according to claim 13 , wherein the mammalian embryonic stem cell is from a mouse.

Assignees

• Suzuki Noriyuki
• Saito Koichi
• Ando Satoshi
• Horie Nobuyuki
• Sumitomo Chemical Co

Inventors

• Suzuki Noriyuki
• Saito Koichi
• Ando Satoshi
• Horie Nobuyuki

Classifications

• C12N5/0603

  Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies · CPC title

• C12Q2600/142

  Toxicological screening, e.g. expression profiles which identify toxicity · CPC title

• A01K2267/0393

  Animal model comprising a reporter system for screening tests · CPC title

• G01N33/5014

  for testing toxicity · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title