Humanized mouse

A technology for mice and immunodeficiency mice, applied in biochemical equipment and methods, embryonic cells, animal husbandry, etc., can solve problems such as difficult to obtain multiple mice

Inactive Publication Date: 2015-02-25
TRANSGENIC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, considering that NOG mice are difficult to breed, it is difficult to obtain multiple mice for experiments

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0227] Establishment of ES cells

[0228] In this example, in order to establish the most suitable humanized mouse for human hepatocyte transplantation, ES cell lines were established from BRJ mouse embryos, and a mouse system was also established.

[0229] (1) Establishment of BALB / c; Rag2- / -; Jak3- / -(BRJ) mice and establishment of ES cell lines

[0230] Rag2-deficient and Jak3-deficient mice were mated to establish double-deficient BRJ mice (Ono A, et al. J Biomed Biotechnol 2011; 539748, 2011. doi:10.1155 / 2011 / 5397481)). The established BRJ mice were used for in vitro fertilization, and 64 blastocyst embryos were obtained, which were cultured in the existing GMEM-KSR medium (14% KSR, 1% FBS, 1000U / ml LIF in GMEM), and tried to establish, But only 2 systems with very poor proliferation can be established.

[0231] These are used to make chimeric mice, but only about 50% chimerism can be obtained, and they do not contribute to the germline. Therefore, CHIR99021, an inhibit...

Embodiment 2

[0241] [Example 2] Induction of mouse hepatocyte death

[0242] (1) Production of a construct for inducing mouse hepatocyte death

[0243] In order to produce a genetically modified mouse capable of specifically killing hepatocytes, two types of constructs were prepared.

[0244] Construct 1 (CAG-ATG-rox-EGFP-rox-DT-A) is connected with EGFP and DT-A (diphtheria toxin fragment A (diphtheria toxin fragment A)) held by ATG and rox immediately downstream of the CAG promoter .

[0245] The initiation codon of EGFP and the ATG upstream of Rox were designed in a fit-in-frame manner. In addition, the initiation codon of DT-A was removed, and the ATG upstream of rox was designed in such a way that it matched the framework.

[0246] Construct 2 (SAP-DreER T2 ) Dre-ER is connected immediately downstream of the promoter of hepatocyte-specific serum amyloid P component (SAP) T2. In addition, a puromycin resistance gene was linked upstream of the SAP promoter.

[0247] The specific ...

Embodiment 3

[0275] Embodiment 3 utilizes the replacement of human growth hormone gene

[0276] Using the homologous recombination vector, exon 1 of the mouse growth hormone (Gh) gene was previously disrupted by the usual method in the same manner as in Example 2. At this time, the ATG of the first exon was destroyed, and a site-specific integration clone with lox71-PGK-beta-geo-loxP-polyA-lox2272 integrated in this part was obtained. Next, a replacement vector is produced. The replacement vector contained lox66-hGH cDNA-polyA-Frt-PGK-puro-Frt-loxP. The replacement vector and the Cre expression vector were introduced into the target recombinant clone by electroporation.

[0277] As a result, an ES clone in which the mouse Gh gene was replaced by the human GH gene was obtained.

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Abstract

The present invention provides embryonic stem cells obtainable from an embryo of an immunodeficient mouse which is deficient in both Rag2 and Jak3 genes by culture in the presence of a GSK3 inhibitor and an MEK inhibitor, as well as a transgenic mouse, which is created with the use of these embryonic stem cells.

Description

technical field [0001] The present invention relates to a mouse obtained by humanizing embryonic stem cells (ES cells) and liver collected from immunodeficiency mice. Background technique [0002] The liver is an organ that plays a central role in the body in terms of metabolism, excretion, detoxification, and maintenance of constant body fluids. The liver is known to be the only organ that regenerates in the living body, and it has the ability to regenerate to its original weight even if about 80% of its total weight is removed. [0003] Since the function of the liver involves many aspects, many genes are expressed in the liver, but correspondingly, there are many genetic diseases caused by the abnormality of the expressed genes in the liver. [0004] When abnormal liver function occurs due to liver disease, etc., there may be no effective treatment other than liver transplantation. Therefore, the necessity of predicting metabolites in human blood increases at the early ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/09
CPCC12N15/09C12N2517/02A01K2207/20A01K2227/105A01K67/0278A01K2267/0331C12N5/0606A01K2207/12A01K67/027A01K67/0271A01K2217/075A01K2267/035C12N5/0603C12N5/0678
Inventor 山村研一荒木喜美冈田诚治下野明彦
Owner TRANSGENIC
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