title: Long-term effects of PERV-specific RNA interference in transgenic pigs.
abstract: 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.
authors.affiliation: Robert Koch-Institute, Berlin, Germany.
authors.forename: Marwan
authors.initials: M
authors.lastname: Semaan
authors.forename: Danny
authors.initials: D
authors.lastname: Kaulitz
authors.forename: Björn
authors.initials: B
authors.lastname: Petersen
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.forename: Joachim
authors.initials: J
authors.lastname: Denner
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D020077:Endogenous Retroviruses / Q000235:genetics* / Q000472:pathogenicity; D015870:Gene Expression; D015751:Genes, pol; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009928:Organ Specificity; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D013552:Swine / Q000235:genetics* / Q000821:virology*; D013997:Time Factors; D014183:Transplantation, Heterologous; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004279:DNA, Viral; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins