RESEARCH ARTICLE Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors for transplantation and pigs are currently favoured as donor animals. However, xe-notransplantation may be associated with the transmission of zoonotic microorganisms. Whereas most porcine microorganisms representing a risk for the human recipient may be eliminated by designated pathogen free breeding, multiple copies of porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated this way. PERVs are released as infectious particles and infect human cells. The zinc finger nu-clease (ZFN) technology allows knocking out specifically cellular genes, however it was not yet used to eliminate multiple integrated proviral sequences with a strong conservation in the target sequence. To reduce the risk of horizontal PERV transmission and to knock out as many as possible proviruses, for the first time the powerful tool of the ZFN technology was used. ZFN were designed to bind specifically to sequences conserved in all known rep-lication-competent proviruses. Expression and transport of the ZFN into the nucleus was shown by Western blot analysis, co-localisation analysis, PLA and FRET. Survival of trans