Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Genome-wide Functional Genetics in Haploid ES Cells
Document related concepts
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genome (book) wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Point mutation wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Oncogenomics wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Transcript
Ulrich Elling Team Leader Institute of Molecular Biotechnology (IMBA) Genome‐wide Functional Genetics in Haploid ES Cells Ourgroupgeneratedhaploidmurineembryonicstemcellsandisdevelopinggenetictoolsforforwardandreverse genomicsapproaches.Thissetupallowsforrapidsaturatingscreenscombiningthepowerofyeastgeneticswith the pluripotencyof embryonic stem cells. In theOmics age highthroughput geneticplatforms are vital bothfor validation of genome wide datasets as well as for the conduction of saturated genetic screens in mammalian systems.RecentgenetictechnologiessuchasRNAiandCRISPRhaveallowedforforwardgeneticscreens;however saturation,offtargeteffects,andreproducibilityarestillpartiallyunsolvedchallengesthatcanbeaddressedinour setup. UsingHaploidGeneticswerecentlyidentifiedmutationsleadingtoresistanceagainstthebioweaponricin(aplant toxin). Such screens are extremely rapid as tens of millions of cells with individual mutations can be assayed in parallelinonedish. Pluripotencyandearlydevelopment Currently,weareusinghaploidgeneticstosystematicallyprobethegeneticframeworkgoverningearlyembryonic development including differentiation, dedifferentiation, reprogramming, lineage decisions, and epigenetic modifications. Which genes are required for ES cell maintenance, which for differentiation? What genes are involvedinlineagedecisionsandcontroloftheepigeneticenvironment?Inordertodoso,weusereporterdriven screening approaches in haploid cells. Analysis of screens is based on massive parallel sequencing of genomic insertionsitesofmutagens. A) Parthenogenesis in mouse oocytes was induced by short exposure to 5% ethanol.Cleavagestageembryosweredevelopedinrecipientfemalesuntilearly blastocyst stage; harvested and embryonic stem cells were derived using standard LIF conditions.Uponseveral FACS sorts,haploidEScellcultures were established. B)Cellsdisplayedahaploidkaryotypeof20chromosomes. C) Immunostaining against ES cell marker genes confirmed ES cell identity, shownareOct4positiveEScellsonaphalloidinlabeledfeederlayer. D) Haploid ES cells differentiate in vitro and in vivo in all three germ layers. Shownarechimericanimalsobtaineduponinjectionofagouti+(brown)haploid EScellsintoBlack6blastocysts. E)Duetotheabsenceofasecondallele,haploidEScellsdisplaylossoffunction phenotypes upon insertional mutagenesis. Lethality or transcriptional reporter systemsareusedtoselectformutationsofparticularphenotypes.Suchscreens aredoneinpoolsofmanymillionindependentmutationsandtherebyveryfast. Mutations in positively or negatively selecting cells are identified using next generationsequencing. F) Haplobank uses pools of mutagenized ES cells to array them and derive cell lineswithidentifiedmutations.Thesecelllinesarearchivedandcanbeusedto testscientifichypothesisderivede.g.fromforwardgeneticscreens. Depletion/DropoutScreens Dropout screens are a standing challenge for functional genomics due to the high demands for signal‐to‐noise ratiosandtheamountofdatarequiredforanalysis.Haploidgenomicsholdsthepromisetoallowforgenomewide depletion screens and we intend to develop it to ask questions beyond what is possible to answer by classical means.Whichmutationsarelethalinsynergywithanoncogeniclesion?Whichmutationshyper‐sensitizeagainst compounds with unknown specificity, i.e. destabilize the drug target pathway? Which genes are required for particularcellstatesandcellularresponses? Collaborations The technology driven nature of the approach fosters multiple interactions and collaborations within and out of campus.Also,ourteaminteractscloselywithHaplobank,anarchiveofmutatedandsequencedconditionalEScell linesthatwehavesetupandrun.Testingcelllinesofinterestharboringspecificconditionalmutationsoutofthe 100 000 lines available cell lines by the end of the year allows for rapid validation of hits identified in genetic screens. Furthermore we are engaged in using haploid genomics for drug target predictions in close contact to industry partners. Further reading Forwardandreversegeneticsthroughderivationofhaploidmouseembryonicstemcells.EllingU,Taubenschmid J,WirnsbergerG,O'MalleyR,DemersSP,VanhaelenQ,ShukalyukAI,SchmaussG,SchramekD,SchnuetgenF,von MelchnerH,EckerJR,StanfordWL,ZuberJ,StarkA,PenningerJM.,CellStemCell.2011Dec2;9(6):563‐74.doi: 10.1016/j.stem.2011.10.012. Haploidembryonicstemcellsandthedominanceofrecessivetraits.,SchimentiJ.,CellStemCell.2011Dec 2;9(6):488‐9.doi:10.1016/j.stem.2011.11.006. Seventy‐fivegeneticlociinfluencingthehumanredbloodcell.vanderHarstP*,ZhangW,MateoLeachI*,Rendon A*,VerweijN,SehmiJ*,PaulDS*,EllingU*etal,Nature.2012Dec20;492(7429):369‐75.doi: 10.1038/nature11677.Epub2012Dec5.*equalcontribution AGWASsequencevariantforplateletvolumemarksanalternativeDNM3promoterinmegakaryocytesneara MEIS1bindingsite.NürnbergSTetal.,Blood.2012Dec6;120(24):4859‐68.doi:10.1182/blood‐2012‐01‐401893. Epub2012Sep12. Newgenefunctionsinmegakaryopoiesisandplateletformation.GiegerCetal.,Nature.2011Nov 30;480(7376):201‐8.doi:10.1038/nature10659. ThestresskinaseMKK7couplesoncogenicstresstop53stabilityandtumorsuppression.SchramekD,KotsinasA, MeixnerA,WadaT,EllingU,PospisilikJA,NeelyGG,ZwickRH,SiglV,ForniG,SerranoM,GorgoulisVG,Penninger JM.,NatGenet.2011Mar;43(3):212‐9.doi:10.1038/ng.767.Epub2011Feb13.
