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Construction method and application of thrombopoietin (THPO) gene humanized non-human animal

A non-human animal, construction method technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of necrosis and apoptosis, immune function deficiency, and weight loss in mice

Active Publication Date: 2021-05-11
BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immune function of NOD-Prkdcscid IL-2rγnull mice is severely defective, there is almost no rejection reaction to human cells and tissues, and a small amount of cells can form tumors (depending on cell line or cell type), and there is no leakage of B lymphocytes, it is the most suitable tool mouse for human cell or tissue transplantation, and has been widely used in the development of new humanized mouse models, but because mouse cells Factors do not act well on human hematopoietic cells, and after transplantation of human hematopoietic stem cells, there are defects in the development and function of human-derived cells
And NOD-Prkdcscid IL-2rγnull mice need to undergo irradiation myeloablation before reconstitution, this procedure requires a radioactive source and will cause multiple side effects in mice , including necrosis and apoptosis of hematopoietic system, gastrointestinal tract, muscle and nerve tissue, which can lead to emaciation, infection and even death of mice, thereby affecting the results of drug efficacy evaluation

Method used

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  • Construction method and application of thrombopoietin (THPO) gene humanized non-human animal
  • Construction method and application of thrombopoietin (THPO) gene humanized non-human animal
  • Construction method and application of thrombopoietin (THPO) gene humanized non-human animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Example 1 THPO Gene Humanized Mice

[0215] In this example, a non-human animal (such as a mouse) is transformed so that the non-human animal contains a nucleic acid sequence encoding a human THPO protein, and a genetically modified non-human animal that can express human or humanized THPO protein is obtained. Mouse THPO gene (NCBI Gene ID: 21832, Primary source: MGI: 101875, UniProt ID: P40226) (based on the transcript of NM_001173505.1 → NP_001166976.1, its mRNA sequence is shown in SEQ ID NO: 1, the corresponding The protein sequence is shown in SEQ ID NO: 2) and the human THPO gene (NCBI Gene ID: 7066, Primary source: HGNC: 11795, UniProt ID: P40225) (based on the transcript of NM_000460.4→NP_000451.1, its mRNA sequence As shown in SEQ ID NO: 3, the corresponding protein sequence is shown in SEQ ID NO: 4) The comparison diagram is shown in figure 1 shown.

[0216] In order to achieve the purpose of the present invention, the gene sequence encoding human THPO prote...

Embodiment 2

[0258] Example 2 Immune reconstitution and verification of genetically engineered humanized mice containing human THPO

[0259] Select 6-week-old THPO humanized mice (B-NDG background, n=20, hereinafter referred to as "THPO mice") and B-NDG mice (n=20), B-NDG mice were irradiated ( 2.0Gy) after myeloablative pretreatment, and non-irradiated THPO mice were injected 1.5×10 5 Human hematopoietic stem cells (HSCs) are used to reconstitute the immune system in mice, and the successful standard of reconstitution is that the ratio of hCD45 to the total living cells is ≥ 25%. Peripheral blood (PB) was taken every four weeks after injection and transplantation for flow cytometry to evaluate whether the reconstruction was successful, observe the state of the mice and record the survival conditions.

[0260] The experimental results showed that at the end of the experiment (the 20th week, specifically the 140th day after injection), the survival rate of THPO mice was 70%, and the surviv...

Embodiment 3

[0266] Embodiment 3 Containing double gene or multigene humanized mouse of human THPO

[0267] Double-gene humanized or multi-gene humanized mouse models can also be prepared by using the method or the prepared THPO 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, fertilized egg cells from CSF2 or IL3 or CSF1 or IL15 gene humanized mice were selected. By performing gene editing according to this method, a double-gene humanized mouse model modified with CSF2 or IL3 or CSF1 or IL15 gene and THPO gene can be further obtained. The homozygous or heterozygous THPO mice obtained by this method can also be mated or fertilized in vitro with other genetically modified homozygous or heterozygous mice, and their offspring can be screened. According to the law of Mendelian inheritance, there is a certain probab...

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Abstract

The invention provides a construction method and application of a thrombopoietin (THPO) gene humanized non-human animal. The method comprises the following steps: introducing a sequence for encoding human THPO protein into an animal genome by using a homologous recombination manner; the animal can normally express the human or humanized THPO protein in vivo, to promote the development of human T cells and NK cells; the non-human animal can be used as an animal model for human THPO signal mechanism research and drug screening for diseases including tumors and the like, and has important application value for new drug research and development of immune targets. The invention further provides a targeting vector of a THPO gene, sgRNA of the target THPO gene and application of the targeting vector and the sgRNA to preparation of a humanized THPO gene.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for constructing a THPO gene-modified humanized animal model based on gene editing and its application in the field of biomedicine. Background technique [0002] Experimental animal disease models are indispensable research tools for the study of the etiology and pathogenesis of human diseases, the development of prevention technologies and the development of drugs. Among them, immunodeficiency animals are easy to accept heterogeneous cells or tissues due to lack of immunity, and have been widely used in tissue or cell humanized animal research, tumor drugs and the treatment mechanism of other diseases. Studies have shown that as recipient mice, the currently commonly used immunodeficiency animals are sorted as follows: NOD-Prkdc scid IL-2rγ nul mouse>NOD-Rag 1 - / - -IL2rg - / - (NRG)>Rag 2 - / - -IL2rg - / - (RG)>NOD...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/19C12N15/113C07K14/52A01K67/027A61K49/00
CPCC12N15/8509C12N15/907C07K14/524C12N15/1136A01K67/0278A01K67/0271A61K49/0008C12N2800/107C12N2310/20A01K2207/15A01K2217/072A01K2227/105A01K2267/0368A01K2267/03A01K2207/12C07K14/575A01K2267/0393A01K2267/0387
Inventor 沈月雷黄蕤郭雅南白阳姚佳维郭朝设张美玲
Owner BIOCYTOGEN PHARMACEUTICALS (BEIJING) CO LTD
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