Construction method and application of CD3e transgenic mouse model for tracing tumor T lymphocyte infiltration

Abstract

The invention provides a construction method and application of a CD3e transgenic mouse model for tracing tumor T lymphocyte infiltration. SgRNA is designed, Cas9 plasmid is taken as a template, the Cas9 plasmid and the sgRNA form homologous recombinant vector plasmid, F0-generation mice are obtained after the homologous recombinant vector plasmid is subjected to fertilized egg micro-injection, F0-generation positive mice are obtained through sequencing, the F0-generation positive mice and wild C57BL / 6J mice are mated, and breeding is carried out to obtain F1-generation heterozygous mice; and selfing of the female F1-generation heterozygous mouse and the male F1-generation heterozygous mouse is carried out to obtain a homozygous progeny, namely the mouse animal model. The humanized animal in which the IRES-EGFP is inserted into the CD3 at the fixed point carries human DNA and carries green fluorescence, so that the infiltration condition of tumor T lymphocytes can be traced, and a research model is provided for tumor micro-environment change of tumor metastasis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of a CD3e transgenic mouse model for tracing tumor T lymphocyte infiltration. Background technique [0002] The prokaryote-derived CRISPR-Cas gene editing system has transformed the ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in living cells of different species. The ease and robustness of the technology has revolutionized genome editing for research from basic science to translational medicine. Based on the principle of the CRISPR / Cas system, relevant personnel artificially synthesized sgRNA sequences in vitro to mediate the recognition and editing of target genes by Cas proteins. Under the guidance of sgRNA, Cas9 is located on a specific deoxyribonucleic acid sequence to cut the double-strand of deoxyribonucleic acid to achieve targeted editing of the genome. The gene editing technology is sim...

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Problems solved by technology

However, there are some limitations and defects. For example, ① using in vitro serially passaged tumor cell lines for transplantation, because the tumor cell lines lack the tumor microenvironment in the in vitro culture environment and lose the characteristics and growth characteristics of the primary tumor, it cannot be objectively It reflects the situation of the primary tumor. ②The transplantation method is often local subcutaneous injection. Local injection of a large number of tumor cells may cause local inflammation and may also limit the microenvironment of the tumor. ③Because the host animal is an immunodeficient animal, it is impossible to evaluate the following Tumor Immunopharmaceuticals Targeting the Immune System

Three humanized animal models currently used for tumor immunotherapy research (reconstructed human immune system animal model, reconstituted human immune system + human tumor tissue xenograft animal model, and genetically modified humanized animal model), (1) Reconstruct the human immune system Humanized mice will develop fatal GVHD at different times after modeling, and the endogenous immune system of mice will limit the expansion of transplanted human cells, etc.

(2) Reconstructing the human immune system + human tumor tissue xenograft animal model Directly transplant fresh human tumor tissue into immunodeficient mice, which has good clinical predictability. It can not only rebuild the tumor immune microenvironment of cancer patients, but also can It can more accurately reflect the tumor status of tumor patients, and has unique advantages in the study of tumor physiology and the preclinical evaluation of targeted drugs, but there are still some limiting factors. Human tumors used for transplantation must be surgically removed fresh tumors, the success rate of transplantation is low, and it takes a long time for tumors to survive after transplantation

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