Urokinase-type plasminogen activator transgenic mouse

Patent metadata

Field	Value
Publication number	US-11051496-B2
Application number	US-201815934520-A
Country	US
Kind code	B2
Filing date	Mar 23, 2018
Priority date	Apr 27, 2012
Publication date	Jul 6, 2021
Grant date	Jul 6, 2021

Abstract

The present invention provides a mouse with liver damage, having a high degree of damage against the mouse's original hepatocytes while having a uPA gene in a heterozygous form, and a method for efficiently preparing the mouse. Specifically, the method for preparing a mouse with liver damage having the uPA gene in a heterozygous form comprises the following steps of:(i) transforming mouse ES cells with a DNA fragment containing a liver-specific promoter/enhancer and cDNA that encodes a urokinase-type plasminogen activator operably linked under the control thereof;(ii) injecting the transformed mouse ES cells obtained in step (i) into a host embryo;(iii) transplanting the host embryo obtained in step (ii) via the injection of the ES cells into the uterus of a surrogate mother mouse, so as to obtain a chimeric mouse; and(iv) crossing the chimeric mice obtained in step (iii), so as to obtain a transgenic mouse in which the DNA fragment is introduced in a heterozygous form.

First claim

The invention claimed is:

1. A chimeric mouse having a chimeric liver that contains transplanted human hepatocytes, wherein:
the chimeric mouse is an immunodeficient mouse having a uPA gene in a heterozygous form;
the mouse does not exhibit rejection against hepatocytes from a different animal origin;
the human hepatocytes were transplanted in an amount between 1 and 2,000,000 cells;
the blood human albumin concentration is 1 mg/mL or higher;
the uPA gene is in the form of cDNA encoding uPA and linked to a liver specific promoter;
and the blood human albumin concentration increases for at least 10 weeks after transplantation of the human hepatocytes,
wherein the chimeric mouse has a replacement rate of human hepatocytes of 60% or more.

2. The mouse of claim 1 , wherein the chimeric mouse was obtained by:
transforming mouse ES cells with a DNA fragment containing a liver-specific promoter/enhancer and cDNA that encodes uPA operably linked under the control thereof;
injecting the transformed mouse ES cells obtained above into a host embryo;
transplanting the host embryo obtained above via the injection of the ES cells into the uterus of a surrogate mother mouse, so as to obtain a chimeric mouse;
crossing the chimeric mice obtained above, so as to obtain a transgenic mouse with liver damage in which the DNA fragment is introduced in a heterozygous form;
crossing the mouse with liver damage with an immunodeficient mouse forming an immunodeficient mouse with liver damage having the uPA gene in a heterozygous form; and
transplanting human hepatocytes into the immunodeficient mouse with liver damage having the uPA gene in a heterozygous form.

3. A chimeric mouse having a chimeric liver that contains transplanted human hepatocytes, wherein the chimeric mouse is an immunodeficient mouse having a uPA gene in a heterozygous form, and the blood human albumin concentration increases for at least 10 weeks after transplantation of human hepatocytes,
wherein the chimeric mouse has a replacement rate of human hepatocytes of 60% or more.

4. The chimeric mouse of claim 3 , wherein the mouse does not exhibit rejection against hepatocytes from a different animal origin.

5. The chimeric mouse of claim 3 , wherein the blood human albumin concentration is 1 mg/mL or higher.

6. The chimeric mouse of claim 3 , wherein the uPA gene is in the form of cDNA encoding uPA and linked to a liver specific promoter.

7. The chimeric mouse of claim 3 , wherein the chimeric mouse was obtained by:
transforming mouse ES cells with a DNA fragment containing a liver-specific promoter/enhancer and cDNA that encodes uPA operably linked under the control thereof;
injecting the transformed mouse ES cells obtained above into a host embryo;
transplanting the host embryo obtained above via the injection of the ES cells into the uterus of a surrogate mother mouse, so as to obtain a chimeric mouse;
crossing the chimeric mice obtained above, so as to obtain a transgenic mouse with liver damage in which the DNA fragment is introduced in a heterozygous form;
crossing the mouse with liver damage with an immunodeficient mouse forming an immunodeficient mouse with liver damage having the uPA gene in a heterozygous form; and
transplanting human hepatocytes into the immunodeficient mouse with liver damage having the uPA gene in a heterozygous form.

8. A chimeric mouse having a chimeric liver that contains transplanted human hepatocytes, wherein the chimeric mouse is an immunodeficient mouse having a uPA gene in a heterozygous form and the blood human albumin concentration is 1 mg/mL or higher and the uPA gene is in the form of cDNA encoding uPA,
wherein the chimeric mouse has a replacement rate of human hepatocytes of 60% or more.

9. The chimeric mouse of claim 8 , wherein the uPA gene is linked to a liver specific promoter.

10. The chimeric mouse of claim 8 , wherein the mouse does not exhibit rejection against hepatocytes from a different animal origin.

11. The mouse of claim 8 , wherein the chimeric mouse was obtained by:
transforming mouse ES cells with a DNA fragment containing a liver-specific promoter/enhancer and cDNA that encodes uPA operably linked under the control thereof;
injecting the transformed mouse ES cells obtained above into a host embryo;
transplanting the host embryo obtained above via the injection of the ES cells into the uterus of a surrogate mother mouse, so as to obtain a chimeric mouse;
crossing the chimeric mice obtained above, so as to obtain a transgenic mouse with liver damage in which the DNA fragment is introduced in a heterozygous form;
crossing the mouse with liver damage with an immunodeficient mouse forming an immunodeficient mouse with liver damage having the uPA gene in a heterozygous form; and
transplanting human hepatocytes into the immunodeficient mouse with liver damage having the uPA gene in a heterozygous form.

Assignees

• Tokyo Metropolitan Inst Medical Science
• Chugai Pharmaceutical Co Ltd
• Phoenixbio Co Ltd

Inventors

• Kohara Michinori
• Jishage Koichi
• Kawase Yosuke
• Mukaidani Chise
• Oshita Hiroki
• Hamamura Satoko

Classifications

• C12N2015/8581

  Animal models for infectious diseases, e.g. AIDS · CPC title

• C12N15/8509 Primary

  for producing genetically modified animals, e.g. transgenic · CPC title

• A01K67/0275 Primary

  Genetically modified vertebrates, e.g. transgenic · CPC title

• A01K2217/052

  inducing gain of function · CPC title

• A01K2227/105

  Murine · CPC title