| Islet transplantation can treat diabetes and maintain blood glucose balance in patients.However,due to the shortage of donor organs and postoperative immunological rejection,it cannot be fully developed.The rejection after surgery is mainly based on T cellmediated cell rejection.And pigs are ideal candidates for islet transplantation donors.LEA29 Y is a synthetic new immunosuppressive agent that blocks co-stimulatory signals between CD28 and B7.1/CD80 or B7.2/CD86,preventing T cell activation.LEA29Y transgenic pigs can suppress T cell-mediated immune rejection in xenotransplantation.However,extensive expression of LEA29 Y results in poor immunity and poor survival of transgenic pigs.Realizing the islet-specific expression of LEA29 Y in transgenic pigs can avoid these problems.It is beneficial to further study the mechanism of LEA29 Y in mammals and provide adequate and reliable donors for islet transplantation to preparation of transgenic pigs regulated by PIP(porcine insulin promoter)and nurture healthy and genetically stable LEA29 Y transgenic pig on islet tissue.We successfully obtained the LEA29 Y primary pig regulated by the PIP(Pig Insulin Promoter)in 2014 and have now been bred to the F2 generation.In this paper,the current islet-specific expression of LEA29 Y pig population was studied.PCR,RT-PCR,Western blotting,immunohistochemistry,and immunofluorescence were used to investigate the identification of genotypes of transgenic pigs and the expression pattern of the target protein.At the same time,we analyzed the stability of LEA29 Y inheritance and expression in transgenic populations.The health status of islet-specific expression LEA29 Y transgenic pigs was evaluated by routine blood tests,paraffin sections,and HE staining.The results show:1.Three LEA29 Y transgenic pigs were positive for LEA29 Y by PCR.A sampling of primary pigs 81 showed that pancreatic specific expression of LEA29 Y can be achieved at the level of transcription and translation.2.Through the natural breeding method,the proliferation of islet-specific expression LEA29 Y pig population can be achieved.The intact and incomplete LEA29 Y vector fragment was integrated into the primary porcine 81 genome,and the incomplete vector fragment could be inherited separately from the offspring.Incomplete vector fragments do not express LEA29 Y and do not affect the expression of intact LEA29 Y.Individuals who inherit only incomplete fragments can be excluded by detecting the integrity of the LEA29 Y sequence.In addition,81 and its hybrids with wild-type pigs inherited the complete destination vector can achieve islet-specific expression of LEA29 Y,inherited stable,can provide a sufficient donor for xenotransplantation.The other 2 primary pigs found non-specific expression of LEA29 Y,and their offspring may be beneficial for transplantation of other organs.3.Islet-specific expression of LEA29 Y had significant effects on blood physiological parameters and immune organs such as thymus and lymph nodes in transgenic pigs.The healthy level of transgenic pigs is favorable to provide a reliable donor for xenotransplantation.In conclusion,through the identification,breeding,screening,and health assessment of islet-specific expression of LEA29 Y transgenic pigs,a genetically stable,healthy,islet-specific expression LEA29 Y pig population was successfully obtained and can provide a sufficient and reliable donor for islet transplantation. |
