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Among all animal species, the pig has the greatest number of anatomical and physiological traits similar to those of humans and thus it seems to be a promising donor of xenogenic organs. The greatest barrier for xenotransplantation in the pig-primate system is connected with the rejection of the transplanted organ via a cascade of immune mechanisms, including hyperacute rejection (HAR), acute humoral transplant rejection, acute cellular rejection and chronic rejection. Many different strategies of genetic modifications in pigs promote adaptation of their tissues and organs to functioning within organisms of their recipients following the transplantation procedure. These changes include e.g. expression of human factors CD39, CD47 and thrombomodulin (TM). Three expression gene constructs were designed and prepared, containing coding sequences for factors CD47, CD39 and human thrombomodulin regulated with the EF- 1α promoter. Gene constructs were used to transfect porcine embryonic fibroblasts by lipofection. Selected cell lines containing individual transgenes were subjected to molecular analysis, which showed integration of introduced transgenes. Cytogenetic analysis showed expression on the protein level in all tested lines and did not detect any chromosomal aberrations in the analyzed transgenic cell lines. Prepared gene constructs were prepared to produce transgenic animals by microinjection. Transgenic cell lines will provide nuclei to produce animals by somatic cloning.
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Author(s): Agnieszka Nowak Anna Woniak Daniel Lipiski Wojciech Juzwa Ryszard Somski Joanna Zeyland
xenotransplantation, transplant rejection, gene construct, domestic pig