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Construction method and application of humanized cell factor IL15 gene modified non-human animal

A non-human animal and genetic modification technology, applied in the fields of plant genetic improvement, chemical instruments and methods, botany equipment and methods, etc., can solve the problems of low differentiation efficiency of human NK and T cells

Active Publication Date: 2020-06-19
BIOCYTOGEN JIANGSU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] But NOD / SCID Il2rg - / - Mice generally lack human T cells and NK cells that can develop normally. When hematopoietic stem cells from umbilical cord blood are implanted, the differentiation efficiency of human NK and T cells is low, especially when human mature peripheral blood is transplanted, human NK cells can only maintain a very short time

Method used

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  • Construction method and application of humanized cell factor IL15 gene modified non-human animal
  • Construction method and application of humanized cell factor IL15 gene modified non-human animal
  • Construction method and application of humanized cell factor IL15 gene modified non-human animal

Examples

Experimental program
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Effect test

Embodiment 1I

[0249] Example 1 IL15 Gene Humanized Mice

[0250] In this example, a non-human animal (such as a mouse) is modified so that the non-human animal contains a nucleotide sequence encoding human IL15 protein, and a genetically modified non-human animal that can express human or humanized IL15 protein is obtained. . Mouse IL15 gene (NCBI Gene ID: 16168, Primary source: MGI: 103014, UniProt ID: P48346) (based on the transcript of NM_001254747.1 → NP_001241676.1, its mRNA sequence is shown in SEQ ID NO: 1, the corresponding The amino acid sequence is shown in SEQ ID NO: 2) and human IL15 gene (NCBI Gene ID: 3600, Primary source: HGNC: 5977, UniProt ID: P40933) (based on the transcript of NM_000585.4 → NP_000576.1, its mRNA sequence As shown in SEQ ID NO: 3, the corresponding amino acid sequence is shown in SEQ ID NO: 4) The comparison diagram is shown in figure 1 shown.

[0251] In order to achieve the purpose of the present invention, the gene sequence encoding human IL15 protei...

Embodiment 2

[0302] Example 2 Double or polygenic humanized mice containing human IL15 cytokine

[0303] Using the method or the prepared IL15 mice can also prepare double-gene humanized or multi-gene humanized mouse models. 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 CSF2 or IL3 or CSF1 gene humanized mice were selected according to this method. Methods Through gene editing, a double-gene humanized mouse model with CSF2 or IL3 or CSF1 gene and IL15 gene modification can be further obtained. The homozygous or heterozygous IL15 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 probability of obtaining...

Embodiment 3

[0304] Example 3 Immune Reconstitution and Verification of Genetically Engineered Humanized Mice Containing Human IL15 Cytokines

[0305] The genetically engineered humanized homozygous mice (B-NDG background, n=15) and B-NDG mice (n=19) of the human IL15 cytokine prepared by 6-week-old embodiment 1 were selected, and irradiated ( 2.0Gy) and then inject 1.5×10 5 Human hematopoietic stem cells (CD34+) 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 collected every four weeks after transplantation for flow cytometry to evaluate whether the reconstitution was successful, and the survival of the mice was recorded. The results of flow cytometry showed that the expression of human leukocyte surface molecular markers (CD45 + ) cells, but the proportion of CD45+ cells in IL15 mice and the number of successfully reconstituted cells were significan...

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Abstract

The invention provides a construction method of a humanized IL15 gene modified non-human animal. A nucleotide sequence encoding a human protein IL15 is introduced into a genome of an animal by using ahomologous recombination mode, so as to construct a gene modified humanized animal. A body of the animal can normally express the human or humanized protein IL15, promote development of human T cellsand NK cells and can serve as an animal model for human IL15 signal mechanism researching and screening of drugs for diseases such as tumors, and thus, the animal has an important application value in research and development of new drugs for immunization target points.

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 humanized animal model of IL15 gene modification based on gene editing and its application in the field of biomedicine. Background technique [0002] Malignant tumors are an increasing threat to human beings. Since the occurrence of tumors is usually latent, and when diagnosed, it has often reached the metastatic stage or advanced stage, so the mortality rate of this disease is still high. Currently, the treatment of cancer mainly relies on surgery, radiotherapy and chemotherapy, but 70% of patients not only fail to achieve the desired effect, but suffer from the side effects of chemotherapy. With the development of life sciences, basic processes such as signal transduction in malignant tumor cells, cell cycle control, induction of apoptosis, angiogenesis, and the interaction between cells and extracellula...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N9/22C12N15/24C12N15/62C12N5/10C12N15/113A01K67/027
CPCC12N15/8509C12N15/907C12N9/22C12N15/1136C07K14/5443A01K67/0278C12N2800/107C12N2310/20C07K2319/00A01K2207/15A01K2217/072A01K2217/075A01K2227/105A01K2267/0325A01K2267/0331C12N2320/11C12N15/11C12N2015/8527A01K67/0276A01K2207/12C12N2830/48A61K49/0008C12N2015/8563C12N2015/8572
Inventor 沈月雷张美玲姚佳维郭朝设郭雅南白阳黄蕤赵磊
Owner BIOCYTOGEN JIANGSU CO LTD
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