Production of Porcine Fibroblasts Harboring MCP Expression Cassette Targeted at Alpha1,3-galactosyltransferase Locus.

Abstract

Xenograft is considered to be an attractive method to solve the severe shortage of organs. However, the major obstacle of pig-to-human xenotransplantation is antibody-mediated immunological rejection, referred as hyper acute rejection and acute humoral rejection which occurs if HAR is prevented. Functional ablation of alpha1,3-galactosyltransferase (alpha1,3-GalT) is the most powerful approach to prevent HAR. Alternatively, overexpression of human complement regulatory protein is known to efficiently decrease HAR and AHR. We developed a vector strategy to lead to disruption of alpha1,3-GalT function and concurrently expression of MCP. We synthesized two homologous region by PCR amplification, corresponding to α1,3-GalT to use 5' and 3' arms for its exon 9. To target separate MCP expression system at exon 9 of alpha1,3-GalT locus, we constructed two MCP expression cassettes, in which CMV and hEF1alpha promoter regulate respectively hMCP expression and IRES-mediated neomycin resistance gene expression. Two cassettes respectively were inserted between 5' and 3' arms (designated as GalT KO/CMV-MCPneo and GalT KO/hEF- MCPneo). The GalT KO/CMV-MCPneo vector was introduced in porcine ear skin fibroblasts using Nucelofector. Nine colonies were identified as targeted at exon 9 of alpha1,3-GalT locus by long-range PCR screening. Quantitative RT-PCR showed that MCP expression is up-regulated, and alpha1,3-GalT is down-regulated. Importantly, we showed that two colonies analyzed expressed efficiently MCP at cell surface. These selected fibroblasts can be used as donor for somatic cell nuclear transfer (BioGreen 21 Program, PJ0071872010). (poster)