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Method for screening optimal gene combination of heterograft donor pig

A technology of gene combination and xenotransplantation, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as blood coagulation disorders

Inactive Publication Date: 2018-07-31
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] A combination of genetically modified pigs that avoid immune rejection, viral infection, and overexpression of certain humanized molecules in porcine vascular endothelial cells to control bioincompatibility effects may address coagulation disorders in xenotransplantation , but there is no conclusion about what gene modification, how many gene combinations, how to combine, and how to evaluate the effectiveness of the gene combination are needed to obtain an ideal donor pig for human xenotransplantation

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  • Method for screening optimal gene combination of heterograft donor pig

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Embodiment 1

[0021] A method for screening the best gene combination of xenotransplantation donor pigs, the specific operation process is carried out according to the following steps:

[0022] 1) Establishment of an in vitro compatibility evaluation platform for human fibroblasts

[0023] Taking human fibroblasts as the experimental object, through human serum-mediated cell proliferation experiment, human serum-mediated cytotoxicity experiment, complement activation test, antibody binding test, complement activity inhibition test and other immunoassay tests, combined with blood coagulation reaction test and lymphocyte activity detection; the average value of each test result of the sample is recorded as 100 points, and the corresponding scoring standards are listed in detail to establish a human fibroblast in vitro compatibility evaluation platform;

[0024] 2) Preliminary establishment of an early and rapid screening platform for the best gene combination of genetically modified xenotrans...

Embodiment 2

[0029] A method for screening the best gene combination of xenotransplantation donor pigs, the specific operation process is carried out according to the following steps:

[0030] 1) Establishment of an in vitro compatibility evaluation platform for human fibroblasts

[0031] Taking human fibroblasts as the experimental object, through human serum-mediated cell proliferation experiment, human serum-mediated cytotoxicity experiment, complement activation test, antibody binding test, complement activity inhibition test and other immunoassay tests, combined with blood coagulation reaction test and lymphocyte activity detection; the average value of each test result of the sample is recorded as 100 points, and the corresponding scoring standards are listed in detail to establish a human fibroblast in vitro compatibility evaluation platform;

[0032] 2) Preliminary establishment of an early and rapid screening platform for the best gene combination of genetically modified xenotrans...

Embodiment 3

[0037] A method for screening the best gene combination of xenotransplantation donor pigs, the specific operation process is carried out according to the following steps:

[0038] 1) Establishment of an in vitro compatibility evaluation platform for human fibroblasts

[0039] Taking human fibroblasts as the experimental object, through human serum-mediated cell proliferation experiment, human serum-mediated cytotoxicity experiment, complement activation test, antibody binding test, complement activity inhibition test and other immunoassay tests, combined with blood coagulation reaction test and lymphocyte activity detection; the average value of each test result of the sample is recorded as 100 points, and the corresponding scoring standards are listed in detail to establish a human fibroblast in vitro compatibility evaluation platform;

[0040] 2) Preliminary establishment of an early and rapid screening platform for the best gene combination of genetically modified xenotrans...

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Abstract

The invention relates to a method for screening an optimal gene combination of a heterograft donor pig and belongs to the technical field of animal biologics. By taking human fibroblast as a study target, an in-vitro compatibility evaluation method for human fibroblast is established, results of reported pig fibroblast modified with heart or other genes with long transplantation survival times canbe verified by using the method, and relatively applicable complement adjustment factors, immune response factors and anticoagulation factors can be screened; on the basis of GTKO (Gene Transferase Knockout), the factors are combined and are subjected to fixed knock-in or knockout, pig fibroblast of different multi-factor combinations is subjected to early-stage screening, then cells of more thanthree optimal gene combinations are recloned, cloned pigs are subjected to heart transplantation of pig-non-primate animals, and the reliability of the screening method can be verified. By adopting the method, an optimal gene combination can be evaluated without the basis that a gene combination modified pig is available, so that the gene combination screening time and the investment cost can begreatly reduced, and the method has great significances in development of real heterograft donor pigs.

Description

technical field [0001] The invention belongs to the field of animal biotechnology, and in particular relates to a method for screening the best gene combination of xenotransplantation donor pigs. Background technique [0002] Organ transplantation, as an effective method to treat patients with end-organ failure, has been widely used clinically. However, this also makes the shortage of human organ donors more and more serious. According to data released by the Organ Procurement and Transplantation Network (OPTN, http: / / optn.transplant.hrsa.gov / data / ) on August 11, 2016, there were 134,559 patients in the United States Waiting for donors, divided by organ type, the number of patients waiting for kidney transplantation is the largest, reaching 109,530 cases, followed by 15,840 cases of liver, 4,088 cases of heart, 2,066 cases of kidney / pancreas, 1,670 cases of lung, 1,084 cases of pancreas, 242 cases of intestine, heart 39 cases per lung; and published organ transplant data s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888
CPCC12Q1/6888C12Q2600/158
Inventor 魏红江赵红业角德灵李鸿辉赵恒
Owner YUNNAN AGRICULTURAL UNIVERSITY
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