Establishment of a strain of haemophilia-A pigs by xenografting of foetal testicular tissue from neonatally moribund cloned pigs

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8(+/−) cloned pigs died of severe bleeding at an early age, as was the case for male F8(−/Y) cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8(−/Y) cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8(+/−) female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8(+/−) cloned pigs, they remained asymptomatic, and delivered five F8(−/Y) and four F8(+/−) pigs after being crossed with wild-type boars. The descendant F8(−/Y) pigs conserved the haemophilia phenotype. Thus, the present F8(+/−) pigs show resolution of the phenotypic abnormality, and will facilitate production of F8(−/Y) pigs as founders of a strain of haemophilia-A pigs for the development of new therapeutics for haemophilia A. This strategy will be applicable to other genetically modified pigs.