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Presenter: Yasuhiro, Takeuchi, London, United Kingdom
Authors: Magda Matouskova1, Pavel Vesely1, Linda Scobie2, Yasu Takeuchi3, Jiri Hejnar1
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CpG hypomethylation as a marker of transcriptionally active porcine endogenous retroviruses
Magda Matouskova1, Pavel Vesely1, Linda Scobie2, Yasu Takeuchi3, Jiri Hejnar1
1Department of Cellular and Viral Genetics, Institute of Molecular Genetics, Prague, Czech Republic; 2Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, United Kingdom; 3Infection and Immunity, UCL, London, United Kingdom
Introduction: The risk of zoonoses is of concern in xenografting human recipients with pig organs, tissues or cells. Porcine endogenous retroviruses (PERV) are able to infect human cells in vitro although no PERV transmission has been recorded in xenograft recipients so far. The porcine genome contains about 50 PERV loci but none of them has unequivocally been associated with in vitro transmission due to their high sequence homology and insertional polymorphism. It is, therefore, difficult to screen for pigs bearing potentially transmissible PERV loci and exclude them from xenograft donors. Provided that CpG methylation inactivates transcription of PERVs, we hypothesise that hypomethylation of PERV long terminal repeats (LTR) might be a marker for such a screening.
Methods: We have developed a quantitative PCR technique (MS qPCR) based on hypomethylation-specific primers universal for all subgroups of PERV LTRs. Bisulfite sequencing was used for independently evaluating the CpG methylation of individual PERV copies or PERV subgroups.
Results: Using the MS qPCR, we examined the level of PERV LTR hypomethylation in tissues and organs of individual pigs from commercial breeds and from the GalKO minipigs. The global PERV methylation shows significant differences among individual pigs although most pig samples tested in this experiment scored one or two orders of magnitude lower than the PK15 cell line, which transmits PERV.
Conclusion: The MS qPCR technique is now ready for screening of xenograft donor pigs.Correlation of PERV LTR hypomethylation with the level of PERV transcription and possible transmission should be further examined. This approach, in combination with other genome-wide analyses, could be also used for identification of PERV loci with potential to produce virus particles transmissible into human cells.
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