A transformed cell based on pig TRAF3 gene knockout and its application in preparing donor for xenotransplantation

Abstract

The application discloses a transformed cell based on pig TRAF3 gene knockout and application of the transformed cell in preparation of a donor for xenotransplantation TRAF3 The application also discloses a gene knockout vector for preparing the donor pig for xenotransplantation TRAF3 The application also discloses a pig TRAF3 After the pig gene is knocked out, the binding capacity of pig tissue and / or organ cells to human IgG and IgM can be effectively reduced, the ability of the pig tissue and / or organ cells to resist human complement-mediated cytotoxicity can be improved, and the survival time of a pig tissue and / or organ recipient can be prolonged. The application can improve the ability of pig vascular endothelial cells to resist human complement-mediated cytotoxicity, so that the pig vascular endothelial cells can be effectively used as a donor animal for inter-xenograft organ and cell transplantation, and a theoretical basis is provided for survival of a subsequent developed xenotransplantation graft.

Description

Technical Field

[0001] This invention relates to the field of xenotransplantation, specifically to a transformed cell based on porcine TRAF3 gene knockout and its application in preparing donors for xenotransplantation. ， preparation TRAF3 Gene knockout pigs are used in xenotransplantation. TRAF3 Porcine vascular endothelial cells can effectively resist human serum-mediated cytotoxicity. Background Technology

[0002] Organ transplantation is an effective clinical treatment for end-stage organ failure and has been widely used in clinical practice. However, its further development is severely limited by the shortage of human organ donors. Xenotransplantation, as a treatment strategy that replaces human organs with organs from other species, holds promise for fundamentally solving the problem of organ donor scarcity if it can be clinically translated, and has become a promising solution of great interest in the field of organ transplantation.

[0003] Numerous studies have confirmed that, among xenotransplantation animal models, miniature pigs share a high degree of morphological similarity with humans in terms of both morphological characteristics and genetic background, making them the most promising source of xenotransplantation organs for clinical application. Yucatan and Göttingen miniature pigs, as the two most widely used experimental animal models, have provided a wealth of valuable experimental data and research conclusions for xenotransplantation-related research.

[0004] However, current research indicates that transplanting miniature pig organs into humans triggers a far more severe immune rejection response than autologous or allogeneic transplantation. This phenomenon is primarily due to the existence of numerous undefined risk factors associated with immune rejection. These factors (including xenogeneic genes that can induce immune rejection) significantly affect the long-term survival of porcine tissues and organs in humans and represent one of the core bottlenecks currently facing the clinical translation of xenotransplantation.

[0005] TRAF3 encodes a protein belonging to the tumor necrosis factor receptor-associated factor (TRAF) family, which can bind to members of the tumor necrosis factor receptor (TNFR) superfamily and mediate their signal transduction. Its core physiological functions include: participation in CD40 signal transduction, contributing to immune response activation; acting as a key component of the lymphotoxin β receptor (LTβR) signaling complex, inducing NF-κB activation and cell death through LTβ binding; interacting with LMP1 encoded by Epstein-Barr virus and other TRAF family members, participating in the regulation of its oncogenic effects; and participating in the regulation of antiviral responses. Furthermore, this protein also possesses protein kinase binding and ubiquitin-transferase activity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for transforming cells based on porcine TRAF3 gene knockout and its application in preparing donors for xenotransplantation. This invention discovers... TRAF3 Genes can cause the risk of xenogeneic immune rejection. This paper provides a transformed cell line with the TRAF3 gene knocked out from a donor pig for xenotransplantation, wherein the donor pig lacks... TRAF3 Genes, pigs TRAF3 After the gene is knocked out, the binding capacity of pig tissues and / or organ cells to human IgG and IgM can be effectively reduced, and the ability of pig tissues and / or organ cells to resist human complement-mediated cytotoxicity can be improved, which has the excellent effect of prolonging the survival time of pig tissues and / or organ recipients.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a transformed cell based on porcine TRAF3 gene knockout, wherein the transformed cell is introduced... TRAF3 Gene knockout vector.

[0008] Furthermore, the pigs mentioned TRAF3 The gene is shown in SEQ ID NO.1.

[0009] Furthermore, the transformed cells are porcine tissue or organ cells.

[0010] Furthermore, the described transformed cell introduction TRAF3 Gene knockout vector. TRAF3 The gene knockout vector includes a vector one, wherein the vector one is linked to an sgRNA, and the sgRNA specifically targets pigs. TRAF3 The sequence from position 1600330 to position 1600349 of the gene.

[0011] Furthermore, the aforementioned TRAF3 Methods for constructing gene knockout vectors: The nucleotide sequence of the encoding gene of the synthesized sgRNA is shown in SEQ ID NO.2, and its complementary strand is shown in SEQ ID NO.3. The single-stranded sgRNA DNA sequences were then annealed to form a targeting... TRAF3 Gene number 1600330 Position 1600349 The oligonucleotide chain of the sgRNA was then inserted into the PX459 plasmid vector.

[0012] The present invention also provides the application of transformed cells in the preparation of donors for xenotransplantation.

[0013] Furthermore, the donor for the xenotransplantation is a pig.

[0014] Furthermore, the present invention sequences the genes of donor pigs or donor pig tissues or organs, wherein the donor pigs or donor pig tissues or organs lack [the necessary genetic information]. TRAF3 Genes can delay, reduce, or prevent rejection, separation, or adverse reactions to xenotransplanted tissues in human recipients.

[0015] Furthermore, the organs mentioned include the liver, lungs, kidneys, or skin.

[0016] Furthermore, the tissue includes nerves.

[0017] The beneficial effects of this invention are: This invention successfully screened and confirmed the presence of pig genome knockout libraries by constructing a whole-genome knockout library. TRAF3 Genes are risk factors for xenogeneic immune rejection; these can be addressed by constructing transformed cells for introduction. TRAF3 Gene knockout vector, knockout TRAF3 Genetic modification can effectively reduce the binding capacity of porcine vascular endothelial cells to human IgG and IgM, and enhance the ability of porcine vascular endothelial cells to resist human complement-mediated cytotoxicity. Therefore, porcine vascular endothelial cells can be effectively used as donor animals for interspecies organ and cell transplantation, providing a theoretical basis for the survival of transplanted xenografts in the future. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a sequencing peak diagram of positive cells with the gene knocked out in the example; Figure 2 The wild pig vascular endothelial cells and in the examples TRAF3 Image showing cell viability of knockout porcine vascular endothelial cells after incubation with human serum; Figure 3 The wild pig vascular endothelial cells and in the examples TRAF3 A graph showing the binding ability of knockout porcine vascular endothelial cells to human IgG and IgM. Detailed Implementation

[0020] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention.

[0021] Example 1: Construction of sgRNA vector.

[0022] According to the NCBI gene bank, pigs as shown in SEQ ID NO.1 TRAF3 Based on the gene sequence, the target region of the sgRNA for gene editing was selected according to the PAM sequence (NGG), and a sgRNA targeting pigs was designed and synthesized. TRAF3 The sgRNA sequence of the gene is as follows: The sgRNA-F sequence is shown in SEQ ID NO.2: 5-TGTGCAGAGCAGTTGACGCT-3; The sgRNA-R sequence is shown in SEQ ID NO.3: 5-AGCGTCAACTGCTCTGCACA-3; The DNA sequences of the above pair of single-stranded sgRNAs were annealed to form a targeted pig... TRAF3 The oligonucleotide chain of the sgRNA at the gene locus; then the sgRNA was ligated into the PX459 plasmid vector to obtain a... TRAF3 Gene knockout vector.

[0023] The sgRNA1-specific targeting pig TRAF3 Gene number 1600330 Position 1600349 A sequence of bits.

[0024] Example 2, Preparation TRAF3 Gene knockout porcine vascular endothelial cells.

[0025] After sequencing verification of the constructed sgRNA expression vector, the target plasmid was extracted and precipitated with ethanol to purify the sgRNA expression vector to a certain concentration. This purified sgRNA expression vector was then introduced into porcine vascular endothelial cells via electroporation. After culturing for 12 hours, the medium was changed, and after 72 hours, gene knockout porcine vascular endothelial cells were obtained through purine pressure selection. The genome of the cells was extracted, and PCR was performed using specific primers. The obtained PCR products were sequenced, and the sgRNA cleavage status of the cells was evaluated by analyzing the sequencing peak diagram. Figure 1 .

[0026] Example 3, Detection TRAF3 The resistance of knockout porcine vascular endothelial cells to human complement-mediated cytotoxicity.

[0027] to be wild and TRAF3 Knockout porcine vascular endothelial cells were washed with PBS, then incubated with human serum diluted in DMEM at a volume ratio of 1:3 for 45 minutes. After discarding the supernatant, the cells were washed twice with PBS and stained with propidium iodide (PI) staining solution for 10 minutes. Cell fluorescence was then detected by flow cytometry (see [link to relevant documentation]). Figure 2Compared to PVEC cells, after incubation with 25% serum, TRAF3 Gene knockout cells can significantly reduce the toxicity of human serum to pig cells.

[0028] Example 4, Detection TRAF3 The binding capacity of knockout porcine vascular endothelial cells to human IgG and IgM.

[0029] Pancreatic enzymes digest wild and TRAF3 Knockout porcine vascular endothelial cells were transferred to 1.5 mL centrifuge tubes and washed twice with PBS. Human serum, inactivated at 56°C for 1 hour, was diluted with PBS at a volume ratio of 1:4 and incubated at room temperature. After 30 minutes, the cells were washed twice with PBS, and a 1:200 volume ratio of fluorescently labeled human IgG or IgM antibody was added to the cells. The cells were incubated at room temperature for 30 minutes. After washing with PBS, the binding ability of TRAF3 knockout porcine vascular endothelial cells to human IgG and IgM was detected by flow cytometry (see [reference needed]). Figure 3 Compared to PVEC cells, TRAF3 gene knockout cells showed significantly reduced binding to human IgG or IgM.

[0030] From the above, we can conclude that: knockout TRAF3 The gene can effectively reduce the binding ability of pig tissues or organ cells to human IgG and IgM, and improve the ability of pig vascular endothelial cells to resist human complement-mediated cytotoxicity. Therefore, the transgenic cloned pig of this invention can be used as a donor animal for interspecies organ and cell transplantation.

[0031] This invention provides a method for preparing transgenic pigs for xenotransplantation, comprising: TRAF3 Knockout pig kidney, lung, or liver cells are transplanted into enucleated oocytes to form nuclear transfer eggs, which are then transplanted into the oviducts of surrogate pigs. Transgenic piglets for xenotransplantation are then born from pregnant sows. These piglets serve as donor animals for interspecies organ and cell transplantation.

[0032] The vector of this invention may contain primer sequences, for example, a CAG promoter. Furthermore, it may utilize promoters commonly considered equivalent to the CAG promoter, such as the EF1α promoter, which are capable of expression in mammals. Additionally, it may use mammalian tissue-specific promoters such as the ICAM2 promoter. The aforementioned CAG promoter, as one type of gene expression promoter, is used to express foreign genes. A "promoter" typically serves as the start of transcription, located in the initial portion of the DNA base sequence carrying the genetic information of the gene to be expressed, within a few hundred bases from the transcription start point. In eukaryotes, proteins called transcription regulators bind to the promoter portion, thereby participating in the binding of RNA polymerase.

[0033] In this invention, "transgenic" refers to the process of introducing DNA into a host and enabling the DNA to replicate as an extrachromosomal factor or through chromosomal integration. Transgenic includes any method of introducing nucleic acid molecules into an organism, cell, tissue, or organ, and can be carried out using appropriate standard techniques known in the relevant field, depending on the host cell, such as electroporation, calcium phosphate precipitation, calcium chloride precipitation, microinjection, polyethylene glycol, DEAE-dextran, cationic liposomes, and lithium acetate-dimethyl sulfoxide, but is not limited to these. To distinguish between transformation of eukaryotic cells using plasmid or non-plasmid naked DNA and transformation as a form of cell tumorigenesis, the term "transfection" is also used, and both have the same meaning in this invention.

[0034] In this invention, the transformed cells are fibroblasts, more preferably porcine fibroblasts, but not limited thereto.

[0035] The present invention provides a method for preparing transgenic pigs for xenotransplantation and transgenic cloned pigs for xenotransplantation produced by the above method, including the steps of transplanting the above-mentioned transformed cells into enucleated oocytes to form nuclear transfer oocytes; and the steps of transplanting the above-mentioned nuclear transfer oocytes into the fallopian tubes of surrogate mothers.

[0036] In this invention, "nuclear transplantation" refers to a gene manipulation technique that artificially combines the nuclear DNA of other cells with a cell without a nucleus to form the same traits, and can use methods known in the technical field to which this invention pertains.

[0037] In this invention, "nuclear transplanted egg" refers to an egg cell that has been introduced or fused with donor cells.

[0038] In this invention, "enucleated oocyte" refers to an oocyte whose nucleus has been removed.

[0039] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A transformed cell based on porcine TRAF3 gene knockout, characterized in that, The transformed cell introduction TRAF3 Gene knockout vector.

2. The transformed cell as described in claim 1, characterized in that: The transformed cells are porcine tissue or organ cells.

3. The transformed cell as described in claim 1, characterized in that, The TRAF3 The gene knockout vector is linked to sgRNA, which specifically targets pigs. TRAF3 Gene number 1600330 Position 1600349 A sequence of bits.

4. The application of transformed cells based on the pig TRAF3 gene knockout as described in claims 1-3 in the preparation of donors for xenograft.

5. The application as described in claim 4, characterized in that, The donor for the xenotransplantation is a pig.

6. The application as described in claim 5, characterized in that, The genes of the donor pig or its tissues or organs are sequenced, wherein the donor pig or its tissues or organs lack [specific genetic information]. TRAF3 Genes can delay, reduce, or prevent rejection, separation, or adverse reactions to xenotransplanted tissues in human recipients.

7. The application as described in claim 6, characterized in that: The organs mentioned include the liver, lungs, kidneys, or skin.

8. The application as described in claim 6, characterized in that: The tissue includes nerves.

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