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2015-04-24Zeitschriftenartikel DOI: 10.1371/journal.pone.0122059
Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN)
dc.contributor.authorSemaan, Marwan
dc.contributor.authorIvanusic, Daniel
dc.contributor.authorDenner, Joachim
dc.date.accessioned2018-05-07T18:13:58Z
dc.date.available2018-05-07T18:13:58Z
dc.date.created2015-05-06
dc.date.issued2015-04-24none
dc.identifier.otherhttp://edoc.rki.de/oa/articles/rel5LLqiJK1AU/PDF/25Q6Tu8BFuDIM.pdf
dc.identifier.urihttp://edoc.rki.de/176904/2058
dc.description.abstractXenotransplantation has been proposed as a solution to the shortage of suitable human donors for transplantation and pigs are currently favoured as donor animals. However, xenotransplantation 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 nuclease (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 replication-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 transfected cells was analysed using fluorescent ZFN and cell counting. After transfection a strong expression of the ZFN proteins and a co-localisation of the expressed ZFN proteins were shown. However, expression of the ZFN was found to be extremely toxic for the transfected cells. The induced cytotoxicity was likely due to the specific cutting of the high copy number of the PERV proviruses, which is also commonly observed when ZFN with low specificity cleave numerous off-target sites in a genome. This is the first attempt to knock out multiple, nearly identical, genes in a cellular genome using ZFN. The attempt failed, and other strategies should be used to prevent PERV transmission.eng
dc.language.isoeng
dc.publisherRobert Koch-Institut, Infektionskrankheiten / Erreger
dc.subjectAnimalseng
dc.subjectHumanseng
dc.subjectGene Knockout Techniqueseng
dc.subjectGene Ordereng
dc.subjectCell Lineeng
dc.subjectProtein Bindingeng
dc.subjectEndogenous Retroviruses/geneticseng
dc.subjectSwineeng
dc.subjectCell Survival/geneticseng
dc.subjectEndodeoxyribonucleases/geneticseng
dc.subjectEndodeoxyribonucleases/metabolismeng
dc.subjectGene Expression Regulation Viraleng
dc.subjectGene Targetingeng
dc.subjectGenomeeng
dc.subjectProviruses/geneticseng
dc.subjectZinc Fingers/geneticseng
dc.subject.ddc610 Medizin
dc.titleCytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN)
dc.typeperiodicalPart
dc.identifier.urnurn:nbn:de:0257-10039421
dc.identifier.doi10.1371/journal.pone.0122059
dc.identifier.doihttp://dx.doi.org/10.25646/1983
local.edoc.container-titlePLoS ONE
local.edoc.container-textSemaan M, Ivanusic D, Denner J (2015) Cytotoxic Effects during Knock Out of Multiple Porcine Endogenous Retrovirus (PERV) Sequences in the Pig Genome by Zinc Finger Nucleases (ZFN). PLoS ONE 10(4): e0122059.
local.edoc.fp-subtypeArtikel
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
local.edoc.container-urlhttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122059
local.edoc.container-publisher-namePublic Library of Science
local.edoc.container-volume10
local.edoc.container-issue4
local.edoc.container-year2015

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