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Construction method of humanized cytokine CSF1 gene modified-non-human animal and application

A non-human animal, genetic modification technology, applied in the direction of chemical instruments and methods, biochemical equipment and methods, and other methods of inserting foreign genetic materials, which can solve problems such as genetic complexity

Pending Publication Date: 2020-04-28
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the differences in physiology and pathology between animals and humans, coupled with the complexity of genes (i.e., genetic factors), how to construct an "effective" humanized animal model for new drug development is still the biggest challenge ( Scheer N, Snaith M, Wolf CR, Seibler J. Generation and utility of genetically humanized mouse models, Drug Discov Today; 18(23-24):1200-11, 2013)

Method used

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  • Construction method of humanized cytokine CSF1 gene modified-non-human animal and application
  • Construction method of humanized cytokine CSF1 gene modified-non-human animal and application
  • Construction method of humanized cytokine CSF1 gene modified-non-human animal and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1 CSF1 Gene Humanized Mice

[0169] Mouse CSF1 gene (NCBI Gene ID: 12977, Primary source: MGI: 1339753, UniProtID: P07141) (based on the transcript of NM_007778.4→NP_031804.3, its mRNA sequence is shown in SEQ ID NO: 1, the corresponding amino acid The sequence is shown in SEQ ID NO: 2) and human CSF1 gene (NCBI Gene ID: 1435, Primarysource: HGNC: 2432, UniProt ID: P09603) (based on the transcript of NM_000757.5 → NP_000748.3, its mRNA sequence is shown in SEQ ID NO: 3, and the corresponding amino acid sequence is shown in SEQ ID NO: 4) The comparison diagram is shown in figure 1 shown.

[0170] In order to achieve the purpose of the present invention, the gene sequence encoding human CSF1 protein can be introduced into the endogenous mouse CSF1 locus, so that the mouse expresses human CSF1 protein. For example, mouse cells can be modified by gene editing technology, at the start codon of endogenous mouse CSF1 (ATG), a coding sequence that can express human CS...

Embodiment 2

[0223] Example 2 Generation of CSF1 and humanized cytokine mice with severe immunodeficiency

[0224] In order to generate mice that contain human CSF1 and have severe immunodeficiency, the CSF1 humanized mouse heterozygote prepared in Example 1 can be mated with B-NDG mice or fertilized in vitro (IVF). The offspring can be screened with a certain probability to obtain heterozygous mice with CSF1 humanization and IL-2Rγ chain deletion, and then cross the heterozygotes to obtain homozygotes with double or multiple genes.

[0225] In the microinjection process in Example 1, the fertilized egg cells of B-NDG mice were used instead of NOD / scid mice to directly obtain B-NDG mice expressing human CSF1 protein.

[0226] The expression of human CSF1 protein in positive mice can be confirmed by conventional detection methods, such as ELISA method and the like. Choose one B-NDG mouse and one B-NDG-hCSF1 homozygous mouse, inject 20μg / 200μL LPS intraperitoneally, take the spleen after 2 ...

Embodiment 3

[0227] Embodiment 3 Contains double gene or multigene humanized mouse of human CSF1

[0228]Double-humanized or multiple-humanized mouse models can also be prepared by using the method or the prepared CSF1 mice. For example, in the aforementioned Example 1, the fertilized egg cells used in the microinjection and embryo transfer process were selected from fertilized egg cells derived from other genetically modified mice, for example, the fertilized egg cells of IL3 or IL15 or CSF2 gene humanized mice were selected using this Methods Through gene editing, a humanized mouse model with double gene modification of IL3 or IL15 or CSF2 and CSF1 can be obtained.

[0229] The homozygous or heterozygous CSF1 mice obtained by this method are mated or fertilized in vitro with other genetically modified homozygous or heterozygous mice, and the offspring are screened. According to the Mendelian inheritance law, there is a certain probability of obtaining CSF1 humanization Digene or polygen...

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Abstract

The invention relates to a humanized gene modified non-human animal, in particular to a construction method of a humanized CSF1 gene modified non-human animal for expressing humanized cytokine proteinand application of the method in the field of biological medicine, and particularly relates to an animal model for expressing human or humanized CSF1 protein. In some examples, a genetically modifiednon-human animal expressing the human CSF1 protein also contains an IL2rg deletion, and / or contains humanization of more genes such as IL3 and CSF2.

Description

technical field [0001] This application relates to the establishment method and application of a humanized CSF1 gene-modified animal model, specifically, to the construction method of a humanized cytokine CSF1 protein-modified animal model and its application in biomedicine. Background technique [0002] The differentiation, development, proliferation and even activation of cells are all subject to the synergistic effect of multiple cytokine signals, among which macrophage colony stimulating factor M-CSF (Macrophage-colony stimulating factor, also known as colony-stimulating factor 1, CSF1) is the bone marrow A cytokine necessary for the differentiation of progenitor cells into monocyte lineage cells, such as macrophages, osteoclasts and microglia, and promotes hematopoiesis in the survival, proliferation, differentiation and maintenance of activity of monocyte-macrophages important role in the function. In addition, studies have shown that CSF1 also plays an important role...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/113C12N15/62A01K67/027A61K49/00
CPCA01K67/0278A01K2207/15A01K2217/072A01K2227/105A01K2267/03A61K49/0008C07K14/535C07K2319/00C12N15/1136C12N15/8509C12N15/907C12N2310/20C12N2800/107
Inventor 沈月雷郭雅南白阳黄蕤尚诚彰张美玲姚佳维郭朝设
Owner BIOCYTOGEN JIANGSU CO LTD
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