Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of α1,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human EF1α promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

Genetic modification of the pig of which the gene is relevant to human diseases allows the pig to be used as a source of biomedical animal model. The promoter which could drive efficient expression constitutively or specifically of the interest gene in porcine organs is essentially required to increase versatility of a biomedical porcine model. In this study, we compared different promoters of activities driving efficient expression in different types of porcine cells including primary fibroblasts, kidney-derived PK-15, and primary endothelial cells (EC). To this end, we inserted CMV, EF1-α, CMV/EF1-α, CAG, human and porcine membrane cofactor protein gene promoters(MCP and Mcp), and porcine intercellular adhesion molecule-2 (Icam-2) promoter into pGL3 basic vector. Luciferase assay revealed that CAG promoter led to highest promoter activity in fibroblasts and PK-15 cells. CMV, EF1-α, CMV/EF1-α promoters showed moderate activities for luciferase expression in fibroblasts and PK-15 cells. Interestingly, CMV/EF1-α promoter, in which CMV promoter was linked to the front of EF1-α promoter as an enhancer led to highest luciferase expression in EC. The MCP, Mcp and Icam-2 promoters showed very low level of luciferase expression in three types of cells. Taken together, this study demonstrated that promoter activity in different porcine cells is differently expressed.

Despite of the absence of hyperacute rejection and acute humoral xenograft rejection, the organ graft of the a1,3-galactosyltransferase (GalT) gene knockouted (KO) and complement regulatory protein (CRP) expressing pig into a nonhuman primate is rejected by development of a thrombotic microangiopathy and/or a consumptive coagulopathy. Thus further introduction of genes to overcome the coagulation incompatibilities between pig and primate under GalT KO/CRP genetic background has been strongly suggested. CD73 (ecto-5'-nucelotidase) is an enzyme attached via a glycosyl phosphoinositol anchor to the extracellular membrane of endothelial cells, which catalyses the hydrolysis of adenosine triphosphate to adenosine. Loss of activity of CD73 results in activation and aggregation of platelets by a reduced capacity to convert nucleotides to adenosine. In previous study, we reported generation of GalT KO fibroblasts concurrently expressing membrane cofactor protein and produced cloned pigs by nuclear transfer of the fibroblast cells (1). In this study, we constructed a vector for expression of human CD73 under control of promoter of pig Icam2 gene expressed specifically at endothelial cells. This vector was introduced into porcine fibroblasts using the nucleofection technology, by which we had forty three fibroblasts clones carrying pIcam2- CD73 vector. Somatic cell nuclear transfer resulted in generation of two transgenic piglets survived.

Immunological rejection of the organ grafted onto a primate arises from two antibody mediated processes, hyperacute rejection (HAR) and acute humoral rejection (AHR). Functional ablation of α1,3-galactosyltransferase (GalT) and concurrently overexpression of complement regulatory proteins are known to inhibit HAR and AHR. In previous study, we reported that production of porcine male fibroblasts harboring a MCP expression cassette targeted to GalT locus. In this study, we constructed a different MCP expression cassette, in which the EF1α promoter regulates MCP expression and internal ribosome entry site-mediated neomycin resistance gene expression. Subsequently, this cassette was inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Female fibroblasts were isolated from ear skin of 10 days old miniature pig, and used for nucelofection of the the construct for MCP expression at GalT locus. PCR analysis showed that four clones of forty neomycin resistant clones carry MCP expression cassette at exon 9 of the GalT gene. Two clones analyzed downregulated GalT expression, as determined by quantitative reverse transcriptase polymerase chain reaction. Flow cytometry analysis showed that MCP was efficiently expressed at the cell surface.

Fibroblasts of large animals are easy to isolate and to maintain in vitro culture. Thus, these cells are extensively applied to donor cell for somatic cell nuclear transfer, and to substrate cells to generate induced pluripotent stem cells after transfection of required genes to be essentially required for direct reprogramming. However, limited mitotic activity of fibroblasts to differentiate along a terminal lineage becomes restrictive for their versatile application. Recently, commercial culture medium and systems developed for primary cells are provided by manufactures. In this study, we examined whether one of the systems developed for primary fibroblasts of human are effective on porcine ear skin fibroblasts. To this end, we performed proliferation assay after five days culture in vitro of porcine fibroblasts in medium DMEM, which is generally used for fibroblasts culture, and medium M106 for human dermal fibroblasts, supplemented with various concentrations of FBS and LSGS contained mainly growth factors, respectively. Consequence was that presence of 15% FBS and 0.1 X concentrations of LSGS in DMEM showed most active proliferation of porcine fibroblasts.