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Establishment method of CTLA4 gene humanized animal model and application thereof

An animal model and construction method technology, applied in the field of animal genetic engineering and genetic modification, can solve the problems of restricting the research and development and application of anti-human CTLA4 antibody, and toxic reactions.

Active Publication Date: 2018-12-18
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at the same time, severe clinical toxic side effects led to the emergency suspension of clinical trials. More than half of the patients treated with Ipilimumab had severe side effects of grade 3-4, leading to toxic reactions in the skin, pituitary and intestinal tract, which limited the ability of anti-human CTLA4 Antibody development and application

Method used

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  • Establishment method of CTLA4 gene humanized animal model and application thereof
  • Establishment method of CTLA4 gene humanized animal model and application thereof
  • Establishment method of CTLA4 gene humanized animal model and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1: Establishment of CTLA4 humanized mouse model

[0049] Using CRISPR Cas9 to replace Exon2 of the human CTLA4 gene with Exon2 of the mouse Ctla4 gene, a CTLA4 gene humanized mouse model was established. C57BL / 6 mice are currently a relatively mature mouse strain, and C57BL / 6 mice were used as background mice to successfully obtain a CTLA4 humanized mouse model.

[0050] (1) Determine the replacement region of the human fragment and the inserted human sequence

[0051] According to the binding functional domain of human CTLA4 and B7 protein, Exon2 of human CTLA4 sequence was selected to replace Exon2 of mouse Ctla4, and the sequence of mouse Ctla4 signal peptide, transmembrane region and intracellular region was retained. The selected human CTLA4 gene sequence is SEQNo. .1 shown.

[0052] (2) Screening of sgRNA prepared from CTLA4 humanized mice

[0053] Design sgRNA targeting murine sequence in the humanized replacement region. Design and synthesize sgRNA s...

Embodiment 2

[0080] Example 2: Expression and functional verification of CTLA4 humanized mouse model

[0081] 1. Verification of mRNA expression in CTLA4 humanized mice

[0082]Detect the transcription of human CTLA4 in heterozygotes by mRNA, and then analyze the expression of humanized CTLA4 gene in heterozygotes and homozygotes by flow cytometry and check the immune cell population. After analysis, CTLA4 humanized mice can be successfully Humanized CTLA4 mice that express the human CTLA4 gene and do not cause obvious abnormalities in the immune system of the mice can be used for subsequent tumor drug efficacy experiments.

[0083] The detection method is as follows: the samples used in the experiment are humanized CTLA4 (KI / wt) and C57BL / 6 wild type (wt / wt) mice. Extract mouse spleen and thymus RNA for RT-PCR, and sequence the PCR products to verify whether the splicing of CTLA4mRNA is correct. Primers (CTLA4-KI-mRNA-F1, CTLA4-KI-mRNA-R1) are located outside the 5-terminus of murine CT...

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Abstract

The invention belongs to the fields of animal gene engineering and genetic modification and particularly relates to an establishment method of a CTLA4 gene humanized animal model and an application thereof. In the invention, the extracellular domain of mouse CTLA4 gene is replaced by a human-source sequence while the intracellular domain holds a complete mouse-source sequence. The CTLA4 gene humanized animal model not only can be used for evaluating the efficacy on tumors of antibodies and has important guiding significance on clinical effect of medicine development, but also is an excellent model for security evaluation on anti-human CTLA4 antibody medicines. The establishment method also includes pre-clinical in-vivo evaluation on the toxicology of the CTLA4 antibody medicine, thus filling market gap.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering and genetic modification, and relates to a method for constructing a CTLA4 gene humanized animal model based on CRISPR / Cas9 technology and its application in biomedicine. Background technique [0002] Complex biological processes often require in vivo analysis, and the study of human in vivo biology is severely constrained by ethics and technology, so animal models are increasingly required for in vivo studies of human cells, tissues, and organs. Currently, scientists have developed a variety of humanized mice or human-mouse chimeras to overcome these limitations and have now become important tools for in vivo studies of human cells and tissues. [0003] During the research and development of clinical drugs, mice are widely used in the preclinical safety and effectiveness evaluation of candidate drugs, such as the in vivo effectiveness evaluation of new antiviral drugs, tumor im...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N15/12A01K67/027
CPCA01K67/0276A01K2207/15A01K2217/075A01K2227/105A01K2267/0331C07K14/70521C12N15/113C12N15/902C12N2310/10C12N2310/20
Inventor 琚存祥赵静马秀英张明坤侯欢欢高翔
Owner GEMPHARMATECH CO LTD
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