BACKGROUND: Porcine endogenous retroviruses (PERVs) represent a risk of 
xenotransplantation using porcine cells, tissues, or organs, as they are 
integrated in the porcine genome and have been shown to be able to infect human 
cells in vitro. To increase viral safety by RNA interference, transgenic pigs 
expressing a PERV-specific small hairpin (sh)RNA targeted to a highly conserved 
sequence in the pol gene (pol2) were generated in which expression of PERVs was 
reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how 
long expression of the shRNA and the RNA interference is effective in reducing 
PERV expression.
METHODS: To analyze the long-term duration of RNA interference, expression of 
the PERV-specific pol2 shRNA and inhibition of PERV expression was studied 
repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of 
transgenic pigs over a period of 3 yr, when animals were sacrificed and 
expression was studied in different organs. Expression of the PERV-specific 
shRNA was measured using a newly developed real-time PCR, and expression of PERV 
was measured using a PERV-specific real-time PCR.
RESULTS: Over a period of 3 yr, PERV-specific shRNA and green fluorescent 
protein (GFP) as reporter of the vector system were consistently expressed in 
transgenic animals. PERV expression was significantly reduced during the entire 
period. Levels of PERV and shRNA expression were different in the various 
organs. PERV expression was highest in the spleen and the lungs and lowest in 
liver and heart. However, in all organs of the transgenic pigs, PERV expression 
was inhibited compared with the vector control animals.
CONCLUSIONS: Transgenic pigs expressing PERV-specific shRNA maintained their 
specific RNA interference long term, suggesting that PERV expression in the 
xenotransplants will be suppressed over extended periods of time.