P225

Establishment of multiple transgenic pig herds for xenotransplantation

Andrea Baehr¹, Nikolai Klymiuk¹, Mayuko Kurome¹, Barbara Kessler¹, Hiroshi Nagashima², David Ayares³, Eckhard Wolf¹

¹Molecular Animal Breeding, LMU Munich, Munich, Germany; ²Laboratory of Developmental Engineering, Meiji University, Kawasaki, Japan; ³Revivicor Inc., Blacksburg, VA, United States

Even though somatic cell nuclear transfer has evolved into a successful method for generating novel transgenic pigs, its low efficiency constitutes a limiting factor in reproduction of already established lines for larger scale experimental purposes. A feasible rectification of this issue may be found in the expansion of transgenic pigs by means of natural reproduction within multiple transgenic breeding herds. The conflicting matters of inbreeding and segregation of multiple transgenes, however, have to be taken into account.

Originating from two GalKO/CD46 transgenic founder boars, one hTM transgenic founder boar and three HLA-E transgenic sows, we have designed a breeding strategy that will lead to a core breeding herd of GalKO/CD46 boars as well as GalKO/HLA-E sows and GalKO/hTM sows over two generations of initial breeding while at the same time allowing for later integration of further transgenes. In order to keep inbreeding within the herd at an acceptable level, regular outbreeding with wild-type pigs is being performed without excessively compromising inheritance of suitable transgene combinations. Integration locus clarification of individual transgenes by inverse PCR allows for determination of transgene zygosity in offspring which aids mating efficiency in terms of expectable transgene combinations in offspring. This has already been accomplished for the hTM transgene while currently being on-going for CD46 and HLA-E transgenes.

Presently matings for pregnancies that will deliver the first F2 generation offspring from which the proposed breeding herd will be recruited are being performed.