Method for carrying out gene modification on non-human animal and constructing immunodeficiency animal model

A non-human animal, genetic modification technology, applied in the field of genetic engineering, can solve the problems of undetectable humanized genes, affect health and development, shorten the life span of mice, etc., achieve a long test window period, good health, avoid Effects on development and physiological function

Pending Publication Date: 2022-01-14
黄菁
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] This application solves the problem of incompatibility of some cytokines in mice and the constructed human immune system, that is, how to express human cytokines in a timely and appropriate manner, because some cytokines that need to be expressed in mice are necessary for the differentiation, maturation, and development of the human immune system. It is required to maintain or exert immunological or physiological related functions, but when it is overexpressed, it will affect the health of mice or shorten the lifespan of mice. If the mouse homologous cytokines are directly humanized to obtain a physiological expression level, The mouse itself will affect the health and development due to the lack of mouse cytokines or because of the tissue specificity of cytokine expression, the expression of humanized genes cannot be detected after humanization of severely deficient mice

Method used

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  • Method for carrying out gene modification on non-human animal and constructing immunodeficiency animal model
  • Method for carrying out gene modification on non-human animal and constructing immunodeficiency animal model
  • Method for carrying out gene modification on non-human animal and constructing immunodeficiency animal model

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Effect test

Embodiment 1

[0147] Example 1 Expression of exogenous genes: human IL3 and human GM-CSF in NOD scid IL2RγKO mice

[0148] 1. Construction of PiggyBac recombinant plasmid (Piggy hCD68-GMCSF / IL3)

[0149] This application utilizes Piggy transposase-dependent transgenic system to overexpress human GM-CSF and human IL3 in mice. Because PiggyBac transposase tends to insert the target fragment into the active transcription region, it will greatly increase the chance of obtaining positively expressed transgenic mice.

[0150] In order to overexpress the gene only in myeloid cells such as monocytes in mice, the vector uses a 3.1kb human CD68 promoter. In order to express different target genes at the same time, the vector uses a double self-cleaving short peptide RAKR-GSG-P2A to connect human IL3 and human GM-CSF genes.

[0151] Piggy transposase-dependent echogenic repeats (ITRs) were designed on both sides of the expression element hCD68Pro-Intron-hGM-CSF-PAKR-GSG-P2A-hIL3-pA. The expression el...

Embodiment 2

[0179] Carry out whole-body gamma irradiation to NVG mice (control group), NVG-hCD68-10-12 mice (experimental group 1) and NVG-SV40-14-X mice (experimental group 2) obtained in embodiment 1 respectively ( 175cGy) to reduce the activity of the mouse-derived immune system. After 24 hours of irradiation, 100,000 individual CD34 stem cells were injected. After 12 weeks, the phenotypes of the human immune system were detected in the mice of the control group and the experimental group. The results were as follows: Image 6 As shown in (A) to (D), after injection of human CD34 stem cells, human lymphocytes, human CD3 T cells, and human CD33 myeloid-derived cells in the experimental group NVG-hCD68-10-12 mice and NVG-SV40-14- The frequency in the blood of X mice was significantly higher than that in the blood of control NVG mice.

[0180] like Figure 7 As shown, the survival rate of NVG-SV40-14-X mice dropped sharply 120 days after injection of CD34 stem cells, and the survival rat...

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Abstract

The application discloses a method for carrying out gene modification on a non-human animal, which comprises the step: carrying out gene modification on a cell fertilized egg of the non-human animal, and carrying out gene editing on the fertilized egg of the non-human animal by utilizing a Piggy transposase dependent transgenic system. The method further comprises the steps: overexpressing a target protein I and/or a target protein II in the non-human animal; the target protein I is a target protein mainly expressed in T cells, B cells and NK cells, and gene expression of the target protein I cannot be detected in severe immunodeficiency non-human animals after gene in-situ humanization of the target protein I; and the target protein II is a target protein which may influence the health of immunodeficient non-human animals. The invention further discloses a method for constructing the animal model with severe immunodeficiency, and a PiggyBac transposon system plasmid. A mouse obtained through the method disclosed by the application is more time-saving and efficient, is better in health condition and has a longer test window period, and the function of the human immune system reconstructed by utilizing the mouse is more complete.

Description

technical field [0001] This application relates to the technical field of genetic engineering, in particular to a method for genetically modifying non-human animals and a method for constructing severe immunodeficiency animal models. Background technique [0002] Humanization of the mouse immune system usually refers to the reconstruction of one or several human immune cells in mice after implantation of human peripheral blood leukocytes (hPBMC) or human hematopoietic stem cells (CD34+HSC) in immunodeficient mice . On the one hand, the humanized mouse immune system can be used to detect the differentiation and colonization of human hematopoietic stem cells and therapeutic cells based on human hematopoietic stem cells. On the other hand, humanized mice can better simulate the human immune system And the tumor immune microenvironment plays an irreplaceable role in many fields such as infectious diseases, antibody drug development, autoimmune diseases and oncology research. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027
CPCC12N15/8509A01K67/0276C07K14/5403C07K14/535C07K14/55C07K14/524C12N2800/90A01K67/027C07K14/54C12N9/14C12N15/85C12N15/90C12N15/89
Inventor 黄菁卢娜
Owner 黄菁
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