Sus artificial chromosomes (SusACs) for the stable segregation of multiple transgenes

• Applicant: Dr. Dirk Schindelhauer
• Subject Area: Cardiac and Vascular Surgery
• Term: from 2007 to 2009
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5470814

In order to delay or prevent organ rejection, pigs with multiple transgenes are to be generated. Difficulties of present transgene vectors include a size limitation (often <10 kb) and inefficient breeding from pigs with multiple loci. Human artificial chromosomes (HAC) have shown that stably segregating low copy elements can be generated de novo by transferring large DMA constructs into human HT1080 tumor cells, provided a centromere proficient satellite DMA was included (>100 kb). However, a de novo satellite sequence has not been cloned in other mammals including mice. The applicant has developed 1) a 50 kb PCR for the subcloning of gene loci, 2) biochemical joining of long DMA constructs and transfer, 3) a telomerized PAC vector (26 kb). The vector contains a CMV/EGFP marker and a white/blue cloning site for genomic library production in E. coli. A satellite enriched library (100-200 kb) will be generated and clones analyzed by sequencing and structural analysis. Centromere formation will be analyzed upon transfer into pig embryonic fibroblasts or mesenchymal cells, and microvascular endothelial cells followed by nuclear transfer in collaboration with Prof Eckhard Wolf in the Forschergruppe. FISH and long term gene expression will be employed to identify pig fetuses with suitable SusACs. The de novo strategy allows the subsequent addition of further genes to the tested constructs to generate next generation organs. The stable expression platform will benefit from pig facilities, the experience with currently expressed genes, and the organ survival and safety analyses by the other members of the consortium.

• DFG Programme: Research Units
• Subproject of: FOR 535: Xenotransplantation