Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

SleepingBeautytransposonmutagenesisinratspermatogonialstemcellsZoltánIvics,1,2ZsuzsannaIzsvák,1,2GerardoMedrano,3,4KarenMChapman3,4&FKentHamra3,4

NatureProtocolsVolume:6,Pages:1521–1535Yearpublished:(2011)DOI:doi:10.1038/nprot.2011.378Publishedonline08September2011

AbstractWedescribeanexperimentalapproachforgeneratingmutantallelesinratspermatogonialstemcells(SSCs)usingSleepingBeauty(SB)transposon–mediatedinsertionalmutagenesis.Theprotocolisbasedonmobilizationofmutagenicgene-traptransposonsfromtransfectedplasmidvectorsintothegenomesofculturedstemcells.Cellswithtransposoninsertionsinexpressedgenesareselectedonthebasisofactivationofanantibiotic-resistancegeneencodedbythetransposon.Thesegene-trapclonesaretransplantedintothetestesofrecipientmales(eitherasmonoclonalorpolyclonallibraries);crossingofthesefounderswithwild-typefemalesallowstheinsertionstobepassedtoF1progeny.Thissimple,economicanduser-friendlymethodologicalpipelineenablesscreensforfunctionalgeneannotationintherat,withapplicabilityinothervertebratemodelswheregermline–competentstemcellshavebeenestablished.ThecompleteprotocolfromtransfectionofSSCstothegenotypingofheterozygousF1offspringthatharborgenomicSBgene-trapinsertionstakes5–6months.

原文：http://www.nature.com/nprot/journal/v6/n10/abs/nprot.2011.378.html