TCR humanized animal model
TCR humanized animal model
TCR humanized animal models, the English name is TCR humanized animal models.At present, there are 1 main methods used for TCR humanized animal models: TCR humanized animal models.
Principle
The basic principle of TCR humanized animal model:
Replacement of mouse-like genes with normal or mutated human genes allows for the establishment of a genetic system or disease model in mice that is closer to that of humans.
The immune system consists of humoral immunity and cellular immunity. Humoral immunity recognizes antigens with antibodies, while cellular immunity recognizes antigens with T cell receptors (TCRs). Both human monoclonal antibodies and T cell receptors can be used as therapeutic molecules.
Appliance
The common application areas of TCR humanized animal models are as follows: (i) Immune system humanized mice have important application prospects in biomedical research and development. (ii) It can be used to generate human monoclonal T cell receptor. (iii) It can be widely used in the research and development of tumor and virus immunotherapy and other important fields.
Operation method
TCR humanized animal model
Principle
(I) TCR-MHC T-cell antigen recognition system: major histocompatibility complex (MHC) molecules can bind with proteasomal or lysosomal processed antigenic peptides of 9-14 amino acid residues, and present the antigenic peptides to the surface of the cell for T-cell recognition; T-cells recognize antigenic peptides presented by MHC molecules through the TCR. HLA is the antigen-presenting molecule in humans, and the MHC molecule in mice is called H2. In the last decade, a deeper understanding of the molecular structure and function of the TCR and the MHC has contributed to the design of better TCR-HLA humanized mice. The antigen processing and presentation systems are fairly conserved between human and mouse cells, with only minor differences, and it is usually not necessary to humanize the genes of the antigen processing and presentation systems, which is conducive to the development of HLA humanized mice. (ii) Structure of human antigen-presenting molecule MHC: MHC molecules are class I and class II. Class I molecules present antigens recognized by CD8 T-cells (peptide 9-11AA), while class 1I molecules present antigens recognized by CD4 T-cells (peptide 14AA). microglobulin (β2m), encoded by the B2M gene. The heavy chain, also known as the a-chain, is a transmembrane protein; the a-chain can be divided into the extracellular membrane region composed of the peptide-binding region and the immunoglobulin-like region, the periplasmic region and the cytoplasmic region. a-chain extracellular membrane region can be subdivided into the peptide-binding region (α1 and α2 structural domains) and the CD8 molecule-binding region (α3). α1 and α2 structural domains are far away from the membrane located on top of the molecule, and their spatial structure is to interact with the antigen and the antioxidant. The α1 and α2 domains are located at the top of the molecule away from the cell membrane and form the spatial structure of the antigen-binding site and the site recognized by the T-cell receptor (TCR). The conformation of the antigen-binding site is in the form of a deep groove, which can accommodate 8-20 amino acid residues, and its size and shape are suitable for antigen fragments that have been processed.The polymorphisms of MHC class I antigen molecules are mainly located in the a-helix structure that forms the sides of the groove, which is related to the function of the class I antigen in presenting antigens; the side chains of the amino acids that form the inner part of the groove are bound to the antigenic peptide mainly through the salt bond and the hydrogen bond. Amino acids located outside and on the surface of the groove are recognized by the TCR; the MHC α3 domain and the β2m protein are located close to the cell membrane, at the bottom of the molecule; the CD8 molecule binds to the α3 domain of the MHC class I molecule; the affinity of the TCR is quite low in comparison to the antibody, and the binding of the CD8 molecule to the α3 domain assists in the recognition of the antigen by the T cell. It is very important to note that the mouse CD8 molecule does not match the human MHCI class of molecules. This means that if the mouse CD8 molecule is used, the human HLA α3 domain should be changed to the mouse α3 domain when designing the HLA transgenic mouse. (iii) Structure of the TCR locus: The TCRα locus (TRA) is located on chromosome 7, and the TCRβ locus (TRB) is located on chromosome 14. In the germline . The DNA encoding the TCR strand is composed of multiple segregated gene fragments; TRA has 127 gene fragments, while TRB has 82-85 gene fragments. Each TCR germline gene consists of gene fragments in the variable (V), binding (J), and constant (C) regions, and in the TCRβ gene, the diverse (D) region. The 5' end of the TCR locus contains different numbers of gene fragments of the V region, which can be divided into subfamilies based on similarity.TRAV genes are subdivided into 29 subfamilies, whereas the TCRβ genes contain 24 subfamilies; the 3' end of the TCR locus contains different numbers of (D)J gene fragments and C gene fragments.TRA contains 61" genes (50 functional); TRB has 2 D genes and 12 J genes. During thymocyte development, the TCR germline genes V(D)J and C gene fragments are rearranged to form a gene encoding a complete peptide chain.
Materials and Instruments
Equipments: PCR instrument, culture medium. Move The basic process of TCR humanized animal model can be divided into the following steps: (I) Process I Caveat The immune system of mice is very similar to that of humans, but the protein sequences of many tumor antigens in mice are different from those of humans, so mouse thymus does not develop tolerance to non-homologous (non-homologous) sequences of human proteins, whereas the knockout mice do not develop tolerance to the entire knockout proteins, thus providing the conditions for the induction and isolation of highly reactive tumor antigen-specific T cells.We have successfully induced tumor antigen immunoreactivity by immunizing TCR-HLA humanized mice with tumor antigen peptide plus CpG and IFA, isolated tumor antigen-specific T lymphocytes such as Melan-A and tyrosinase, and then prepared the cDNA of their TCR.The TCR cDNA sequences were then cloned into retroviral vectors. These TCRs were experimentally confirmed to recognize tumor antigens. Transfection of primary human T-lymphocytes with these TCR vectors transformed these cells into anti-tumor T-lymphocytes that could kill target cells. For more product details, please visit Aladdin Scientific website.
Reagents: relevant genetic engineering enzymes, artificial yeast.
B. Mate with third-generation HLA-A2 (HLA, human leukoeyte antigen human leukocyte antigen) transgenic mice and Db/β2m knockout mice ( HHDII strain).
C. Then mated with TCR-α/-β knockout mice to produce humanized T cell antigen recognition system mice.