Background: The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post-transcriptional gene expression, offers a promising alternative to achieving this goal.
Methods: Here, the combination of nuclear transfer cloning and RNAi technology was used to produce pigs that may not propagate PERV. Small interfering RNAs (siRNA) were expressed as short hairpin RNAs (shRNA) against the gag and pol PERV genes, respectively, under the control of a RNA polymerase III (pol III), or a pol II promoter. PERV gag and pol model-genes, in combination with a Green Fluorescent Protein (GFP) reporter system, were developed to assess in vitro PERV target knockdown. Two shRNAs were selected, and transgenic pigs were produced that expressed the anti-gag and -pol shRNAs, in tandem, under the control of a ubiquitous pol II promoter.
Results: The anti-gag and -pol shRNAs, effectively knocked down expression of the PERV model-genes, and also endogenous PERV within cells in vitro. PERV knockdown was achieved whether the shRNA was expressed under the control of a RNA pol III, or a pol II promoter. Three litters of cloned pigs were produced. The shRNA construct was expressed by all the transgenic cloned animals, and within all the tissues of transgenic animals tested. PERV expression at the mRNA and PERV particulate levels in the pigs was virtually undetectable, compared with the infectious levels expressed by the positive control PK15 cell line in vitro. Immunofluorescence and Western blotting, with an anti-PERV-envelope antibody, did not detect PERV in pig tissues or cells whether activated or not, as compared to the positive control on PK15 cells.
Conclusions: The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.