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Organ-humanized mouse

A mouse and human technology, applied in biochemical equipment and methods, artificially induced pluripotent cells, embryonic cells, etc., can solve problems such as difficulty in obtaining multiple mice and difficulty in breeding NOG mice.

Inactive Publication Date: 2018-02-16
TRANSGENIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, due to the difficulty of breeding NOG mice, it is difficult to obtain as many mice as needed for experimental use

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] Establishment of ES cells

[0233] In this example, in order to establish the most suitable humanized mice for human hepatocyte transplantation, ES cell lines were established from HHB mouse embryos, and mouse strains were also established.

[0234] (1) Establishment of HHB mice and establishment of ES cell lines

[0235] HHB mice were used for in vitro fertilization to obtain 33 blastocyst embryos. CHIR99021, an inhibitor of GSK3, and PD0325901, an inhibitor of MEK, which are effective for maintaining the undifferentiated state of ES, were added to the medium (GMEM- KSR-2i medium), try to establish ES.

[0236] Specifically, embryos of HHB were collected by in vitro fertilization. 33 blastocysts were cultured on KSOM medium for 4 days until embryo sacs formed, and 1 embryo was placed in each of 48 wells (coated with gelatin only). The medium used was KSR-GMEM-2i medium. As the medium composition, G-MEM (Glasgow minimum essential medium) contains 1X MEM non-essentia...

Embodiment 2

[0244] Induction of mouse hepatocyte death

[0245] (1) Production of a construct for inducing mouse hepatocyte death

[0246] In order to produce genetically modified mice capable of specific disappearance of hepatocytes, two constructs were produced.

[0247] Construct 1 (CAG-ATG-lox-EGFP-lox-DT-A) had ATG, lox-clamped EGFP, and DT-A (diphtheria toxin fragment A) linked immediately downstream of the CAG promoter.

[0248] The start codon of EGFP and the ATG upstream of ROX were designed in frame fit. Additionally, the start codon for DT-A was removed and designed in such a way that it fits in the ATG framework upstream of rox.

[0249] Construct 2 (SAP-CreER T2) is linked with Cre-ER immediately downstream of the promoter of hepatocyte-specific serum amyloid P component (SAP) T2 . Furthermore, a puromycin resistance gene was linked upstream of the SAP promoter.

[0250] The specific method is as follows.

[0251] (1-1) Construct 1

[0252] Construct 1 was made as fol...

Embodiment 3

[0280] Human Growth Hormone Gene Replacement

[0281] Using a homologous recombination vector, exon 1 and exon 2 of the mouse growth hormone (Gh) gene were pre-destructed in the same manner as in Example 2 by conventional methods. At this time, the ATG of the first exon was destroyed, and the target recombinant clone in which lox71-PGK-beta-geo-loxP-poly A-lox2272 was embedded was obtained. Then make the replacement vector. The replacement vector contains lox66-genomic hGH gene-polyA-Frt-PGK-puro-Frt-loxP. This replacement vector was introduced into the target recombinant clone by electroporation together with the Cre expression vector.

[0282] As a result, an ES clone in which the mouse Gh gene was replaced with the human GH gene was obtained.

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Abstract

An embryonic stem cell that is obtained by culturing a mouse embryo, in which the whole or a part of domains of H2-D molecule of mouse MHC class I are substituted by domains of HLA-A molecule of humanMHC class I, in the presence of a GSK3 inhibitor and an MEK inhibitor; and a mouse that is produced using the embryonic stem cell.

Description

technical field [0001] The present invention relates to embryonic stem cells (ES cells) and organs collected from mice in which all or part of the domains of the H2-D molecules of the mouse MHC class I are replaced with the domains of the human MHC class I HLA-A molecules Humanized mice. Background technique [0002] In the past, regarding the making of model mice of human organs, for example, for the model mice of the liver, Heckel et al. reported a transgenic mouse (Tg(Alb-Plau), which was introduced with a construct (Alb-Plau), Said construct is linked with the plasminogen activator (Plau) gene of urokinase (urokinase) type on the albumin (Alb) promoter (non-patent document 1: Heckel et al. Cell 62:447-456,1990 )), however, this mouse died on the fourth day after birth due to hemorrhage of the intestinal tract, etc., and thus was not used in the experiment. On the other hand, in Tg(Alb-Plau), the system establishment of surviving individuals was successful, and an examp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027
CPCC12N5/067C12N5/0671C12N2501/115C12N2501/12C12N2501/16C12N2501/235C12N2501/237C12N2501/39C12N2501/727C12N2506/45C12N2510/00C12N2517/02A01K2267/02C07K14/70539A01K67/0278A01K2217/075A01K2227/105C12N5/0606A01K2267/03C12N2501/998
Inventor 山村研一李正花
Owner TRANSGENIC
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