Systematic design,production and breeding of multi‐transgenic donor pigs

The continuously increasing shortage of donor organs for allotransplantation demands the development of alternative treatments. The implementation of xenotransplantation suffers from various pathways of rejection and incompatibilities between recipient and donor tissues. Usage of transgenic donor pigs might overcome these problems, but the generation of transgenic animals by nuclear transfer is costly and teadious, the combination of several transgenes represents considerable efforts and the permanent production of donor pigs requires optimization between the conflicting issues of transgene segregation and high inbreeding coefficient. The establishment of novel transgenes is normally biased, which means that with increasing knowledge on the diverse mechanisms of xenograft failure, novel transgene variants arise. In addition, the poor knowledge about the porcine genome and site‐specific regulation in pigs requires concise planning and pre‐evaluation of bio‐informatic data. The generation of transgenic pig follows the conventional and well established strategies in mouse, with the exception that no embryonic stem cells are available in the pig. But the establishment of transgenic primary cells, their usage as donors for somatic cell nuclear transfer and the transfer of cloned embryos into synchronized gilts represent a reasonable alternative, conventionally resulting in suitable founder animals within a few litters. The characterization of the founders depends on the site of expression and, thus, influences the strategy of founder reproduction. The lateron combination with other transgenes is challenging and normally requires implementation of the transgene into an existing breeding herd. Thus, the breeding concept has to be designed to (i) to establish a core herd, (ii) to allow the addition of novel transgenes to the herd and (iii) to support the maintainence of the breeding herd by continuous re‐juvenation. The latter is of certain importance, as transgenes normally segregate according to the Mendelian pattern of inheritance. This aspect would favour a homozygous transgene status for all breeding animals. However, such a strategy will result in dramatically increasing inbreeding coefficients and, consequently, reduce fertility and litter sizes. Taken together, both, the permanent production of multi‐transgenic donor animals as well as the development of novel transgenes and their integration into a breeding herd is a logistic challenge that requires long‐term planning but also flexibility regarding the transgene combination which depend is governed by the increasing insight in the diverse mechanisms of xenograft failure.