1 issue 4 2016 phenogenomics

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NEWSLETTER

Volume 1 Issue 4 2016

PHENOGENOMICS

Czech Centre for PhenogenomicsCzech Centre for Phenogenomics

Participants: Amaximumof15participantsincluding2scholarships.

Application deadline: 15thFebruary2017(resultsposted20thFebruary2017)

Course fee: 980Euro*

Participationonthecourseissubjecttoaselectionprocess.ApplicationsmustbeaccompaniedbyashortCV,letterofmotivation(max200words),andashortletterofsupport.Participantswishingtoapplyforthescholarshipshouldstatethisclearlyinthecoverletter.Pleasenotethatthescholarshipwillcoverthecoursefeeonly.

*Thisfeeisforparticipantsfromacademicinstitutions.Thecostforparticipantsfromindustryisavailableonrequest.

Thiscourse,jointlyprovidedbytheCzechCentreforPhenogenomicsandPhenomin/ICS,bothmembersofINFRAFRONTIER,isdesignedtogivepractical,hands-ontrainingincurrenttechnologiesandmethodsemployingCRISPR/Cas9-mediatedgenomemanipulationsinthegenerationofmicemutants.Participantswill gain theoretical and practical knowledge fromour expert instructors, have ample opportunity fordiscussionandcanbringtheirowntargetingprojectstoworkonduringthebioinformaticssession.Thecoursehasbeendesignedforparticipantswithanintermediateknowledgeofmolecularbiology.

Coursehighlights:

• Designtargetingstrategyforyourprojectofinterest

• ApplythetargetingtoolsandvalidateCRISPRactivity

• Delivertheeditingtoolstocelllinesandzygotes

• IdentifyCRISPRinducedgenomicmodifications

Czech Centre for Phenogenomics

2nd Programmable nucleases (CRISPR/Cas9)

Transgenesis Course

3rd-7thApril2017

CzechCentreforPhenogenomics,Prague-Vestec(BIOCEV)

Applicationsshouldbesenttothefollowingemailaddress:[email protected]

For more information visit our website: www.phenogenomics.cz

VOL. 1 ISSUE 4PHENOGENOMICS NEWSLETTER 3

Contents

Editor NicoleChambers

Editorial teamInkenM.BeckKarelChalupskyKallayaneeChawengsaksophakTrevorEppIvanKanchevAgnieszkaKubik-ZahorodnaJiriLindrovskyPeterMakovickySilviaPetrezselyovaBenoitPiavauxJanProchazkaMilanReinisRadislavSedlacekSarkaSuchanova

Printers: AMOS typograficke, Praha 4

Cover Image:LacZstainingoflungsshowingspecificgeneexpressionin thebronchialtree.

Photo CreditsChristopherChambersAgnieszkaKubik-Zahorodna

Note to customers:Asvaluedcustomers,wewelcomeyourarticlesandfeedbackontheserviceyoureceived.Pleasesendallcorrespondencetoccp@phenogenomics.cz

Theeditorialteamwouldliketothanktheauthorsinthisissuefortheircontribution.

NewsInBrief

DayforNationalResarchInfrastructures 5

BioanalyticswithournewQ-TOF-MSinstrumentation 5

FeaturedReview

EmbryonicStemCellTargetingTools 6

AnOverviewofprogrammablenucleasetechnology 9

FeaturedService

MetabolismUnit 12

FeaturedReview

Lungfunctionmeasurementinlaboratoryanimals: Anhistoricaloverview 14

IntheSpotlight

GoodScienceneedsGoodModels 16

ShorthistoryofPWD/PhandPWK/Phmouseinbredstrains 18

Careers 22

UpcomingEvents 23

JournalClub 23

4PHENOGENOMICS NEWSLETTER VOL. 1 ISSUE 4

Czech Centre for Phenogenomics, Institute of Molecular Genetics

offers

FREE-OF-CHARGE SERVICE OF HISTOPATHOLOGY UNIT

The Czech Centre for Phenogenomics (CCP), Institute of Molecular Genetics ASCR (CCP-IMG) supports researchers with a free of charge service of histopathology unit. Atotalof5 histopathology projects willbeperformedforfree.

Theservice providedcovers:

• Organ sampling and trimming

• Tissue processing

• Embedding

• Sectioning

• H&E staining

• Slide scanning

1project:1micecohort(max15mice,5organspermouse)orequivalentnumberoftissues

Theusermustworkinanacademicinstitution

Servicerequestsforthiscallshouldbemadeviaemailccp-pm-histo@img.cas.cz withsubject“Callforfree-histopathology”).

Allrequestswillbesubjecttoareviewprocedure,whichwillbeinitiatedafterthecallisclosed.Applicantswillbeinformedontheoutcomeoftheevaluationwithin3weeksaftertheendofthecall

Call,startingdate:1stJanuary,2017

Call,closingdate: 31stJanuary,2017


Section Title

Karel ChalupskyClinical Chemistry Unit

Libor DanekCCP Management

Bioanalytics with our new Q-TOF-MS instrumentation

Day for National Research Infrastructures

News In Brief

AfterwecantakecareofgenomeandtranscriptomeviaCrispr/Cas9technology,themetabolomeisnowinoursight.Metabolomeisduethemicrobiomeandenvironmentalinfluencestheonly–omethatdoesnotrelycompletelyonDNAandproteinexpression,therefore its changesmore reflects fast changes in metabolism, diet and life style.Recently,wehaveinstalledanewQ-TOF-MSinourcenter,andthisnewinstrumentationenablesustooffermoredatainlesstimewhenusedforthebioanalyticservices.UsingtheMETLINMetabolomicsDatabase,whichisarepositoryofmetaboliteinformationaswellastandemmassspectrometrydatatogetherwithKEGGdatabase,themetabolitesaremined,identifiedandlinkedwithmetabolicpathways.Wewanttoaddnon-targetedmetabolomicsinourregularpipelinewherewesearchfornewgenefunctions.WeplantouseQ-TOFbesidesclassicalclinicalbiochemistryscreenbutinordertosafesamplevolumewemightswitchandvalidateQ-TOFapproachforsomeclinicallyusedmetabolitesinfuture.Additionallyalsowewanttoimplementtargetedmetabolomicsformetabolitessuchbileacidsandsteroidshormonesinourresearchprograms.

On Thursday November 3 2016 the representatives of almostsixty Czech research infrastructures, research policy agencies,stateadministrationandotherstakeholdersmetforthefirsttimetosharetheirviewsandexperienceduringtheNationaldayofresearchinfrastructures,uniqueconferencejointlyorganizedbyELIBeamlines,CCP/BIOCEVandIT4IonNovember3rd,2016inDolníBřežany.

Theresearchinfrastructures(RI)areuniquefacilitiesandresourcesofferingvarioustop-levelservicesforresearchcommunitiesonnationalandinternationallevel.WithintheEUtheEuropeanStrategyForumonResearchInfrastructures(ESFRI) coordinatesacoherentandstrategy-ledapproachtopolicy-makingonRIinEurope.OneofthekeyoutputsofESFRIisregularlyupdatedroadmapof theRI that identifiestheRIofpan-European interestcorrespondingto the longtermneedsof theEuropeanresearchcommunities.ThesimilarnationalroadmapshavetobeproducedbyEUmemberstates.

AlbeittheRIintheCzechRepublicarerepresentedbytheRICouncilservingasadvisoryboardtoMinistryofEducation,YouthandSports,thenationaldayofRIinBřežanywasthefirsteventwhererepresentativesofallCzechresearchinfrastructureshavetheopportunitytomeetnotonlythemselvesbutalsoallrelevantstakeholders(e.g.MinistryofEducation,GovernmentOfficeforResearch,AcademyofSciencesetc.).TheESFRIwasrepresentedbyitschairprof.GiorgioRossiandhisdeputyDr.JanHrusak.

The key topics that participants discussed in the first part of the conference were the necessary conditions and favorableenvironmentneededforoperatingnationalRIasbackboneofexcellentresearchintheCzechRepublic.Obviouslythesustainabilityandfundingarekeyissuesformostoftheinfrastructures.TheinternationaldimensionofsuchissueswerementionedbyESFRIrepresentativeswhospokeonnewESFRIroadmapfor2018andlong-termsustainabilityofRIinEUpolicyrecommendations.TherepresentativesofMEYSandCzechGovernmentstakeallthatinconsiderationforfurtherdevelopmentofnationalRIpoliciesandresearchfinancingbudgetproposals.

Thefollowingpaneldiscussionswerededicatedtomutualexchangeofviewsondailyroutineoperationof infrastructuresandservingtheusers.CzechresearchersarenotyetenoughusedtoutilizetheservicesofRIandisoneoftheimportanttasksofRItoattractthem.OntheothersidetheinfrastructureshavetofollownationalandEUprovisionsoncompetitivenessandstateaidandthusmightbelimitedincommercialprovidingofcontractedresearchservices.TheotherconcernsarerelatedtoopenaccesspolicywheretheindividualRShavequitedifferentapproachregardingscopeoftheservicesoffered,selectingtheusersorhowtosharecollectedresearchdata.NonethelessallparticipantsagreedthatusersatisfactionhavetobekeybenchmarkfortheRI.


AttheTransgenicandArchivingModule(TAM)ofCCP(https://www.phenogenomics.cz/)we producemousemodels for theIMPC(InternationalMousePhenotypingConsortium)byusingtargetedembryonicstemcells(ESCs)aswellasprogrammablenucleases(CRISPR/Cas9system).TheproductioniscompletelyonC57Bl/6Nbackground,sinceitisoneofthebestcharacterizedinbredstrainsandservesasreferenceforthesequenceofthemousegenome.

This article would like to give an overview about differentavailable alleles that are mainly used for targeting genes inESCsinframeoftheIMPC.ThetargetedESCsallelesdescribedherearemainlyavailableviatwomajorrepositories:EUMMCRinMunich (EuropeanMouseMutant Cell Repository, https://www.eummcr.org/) andKOMPat theUniversityofCalifornia,Davis(NationalInstitutesofHealthKnockoutMouseProgram,https://www.komp.org/index.php). Over ten different allelesweredevelopedunderIKMCthatalsocomprisesthreefurther

programs,TIGM,NorCOMMandEUCOMMTOOLS.

Withtheaimofgeneratingaresourceofreporter-taggednullmutation in mouse ES cells, large-scale knockout consortia1 were established to support and improve efforts towardsthe genetic analysis of all mammalian genes https://www.mousephenotype.org/about-ikmc.ThegoaloftheInternationalMouse Phenotyping Consortium is to discover functionalinsight foreverysinglegenebygeneratingandsystematicallyphenotyping 20,000 knockout mouse strains (http://www.mousephenotype.org/).

The high through-put pipeline developed at the WellcomeTrust Sanger Institute2 forms the basis for the generation ofthousandsoflacZ-taggedconditionalalleles.Conditionalallelesprovide the possibility to study gene function in tissue- andtime-specific manner. Main and most versatile IKMC alleleis the so called “knockout-first” allele that combines a LoxP-flanked critical region togetherwith lacZ element terminatedby polyadenylation sequence (see Figures). Promoterless andpromoter-driventargetingcassettesareinusetogeneratethisknockout-first allele and in contrast to classical conditional

alleles, this allele aims to already generate a null mutation.By using site-specific Flp and Cre recombinases conditionalwild-type and reporter knockout alleles, respectively, can beproduced.

The pictures show the different alleles that can be derivedfrom original tm1a allele. By using Cre recombinase cuttingat loxP sites the critical region will be removed completelyand generates the tm1b allele. For screening mice throughIMPC phenotyping pipelines mice carrying the tm1b alleleare taken. The tm1a version with promoter-driven selectioncanbeconvertedtoatm1ballelewithoutneomycincassette. Insomecases,ithasbeenreportedthatpresenceofneomycinresistancegeneinfluencesphenotyperesults.

Togenerateaclassicalconditionalallele(tm1c)Flprecombinasecutting at Frt sites is needed to excise lacZ reporter andselectionsequences,resultinginawild-typeallelewithfloxedcriticalregion.Thistm1callelecanfurtherbetreatedwithCrerecombinase to generate tissue-specific deletion or deletionin time-specificmanner in presence of inducible Cre protein(tm1dallele).

For the conversion into tm1b and tm1c allele, with highefficiencyCCPisusingCreandFlpdeleterstrains,respectively,that were developed and generated at ICS, Strasbourg3. Anelegant,timesavingandefficientmethod to convert tm1aallelestotm1bwasestablishedbyusingacellpermeableCrerecombinasetreatmentofpre-implantationembryos4.

Inken M. BeckTargeting and Archiving Module

Embryonic Stem Cell Targeting For IMPC

Featured Article

Fig.1Knockout-firstallele:Promotorlessselectioncassette

Fig.2Knockout-firstallele:Promotor-drivenselectioncassette

Fig.3Targeted,nonconditionalallele


During targeting in ESCs the 3’ loxP site is often lost due torecombination events in the homology arms between thetargetingcassetteand3’loxPsite.Thisgeneratestargeted,butalleleswithoutconditionalpotential(tm1e),meanstheycannotbe transformed to conditional alleles using Flp recombinase.TheallelesarelacZ-taggedandshouldresultinnullmutations(Fig3).

TheVelociGenegroupatRegeneron,Inc.generatedadifferentgeneral construct for creating knockout alleles for KOMP (Fig4). Inmost cases the targetingwill result in a complete nullallele(tm1)thatdeletestheentireproteincodingsequenceofthetargetgene.ThisalleledesigncanbeappliedtoanygenetranscribedbyRNApolymeraseIIregardlessofsize,intron-exonstructure, RNA splicing pattern, or protein-coding capacity5. For IMPC phenotyping efforts selection cassette has to beremoved by Cre recombinase leading to a reporter-taggeddeletionallelewithoutselectioncassette(tm1.1).

Detailed information about nomenclature for mutant allelesgenerated in ES cell lines by IKMC can be found at MouseGenomeInformatics(MGI)websiteunderfollowinglink:http://www.informatics.jax.org/mgihome/nomen/IKMCnomen.shtml

TheaimoftheIMPCistoprovidedataonC57Bl/6Nbackground,

accordingly,alsotargetedESCsusedforIMPCproductionareallderivedfromthisinbredstrain.TherepositoriesuseddifferentB6NESCs lines to target the genes. Pettittet al. developedavaluableESC line, JM8,withhighgermlinetransmissionrate6.Byconvertingthenon-agouti(a/a)mutationbacktodominantagouti(A/a)allele,sublinesweregeneratedcarryingtheagoutifurcoloroverblackfurcolorinformation.

By injecting these A/a ESCs into a/a embryos it allowsvisualization of ESC-derived mice by coat color and ensuresrecoveryofpureinbredmicefromtestcrossesperformedwithC57BL6/Nmice.

AnotherESClinewithreliablegermlinetransmissionefficiencyusedforhigh-throughputgenetargetingisVGB6thatismainlyused by VelociGene group for generation of tm1 deletionalleles7.

Tomakebenefitofcoatcoloridentificationforthegenerationofchimericmice,ourmoduleisusingC57Bl/6NandC57BL/6J-Tyrc-2J(albinoB6)embryosashoststrainsforinjectingtheESCs.Withthisdonorstrainswehaveopportunitytoinjecta/aandA/aESCsandalsoESCsderivedfromdifferentbackgroundswithpotential to see if and howmuch the stem cells contributedtothechimerism.AstestcrossstrainforchimerasinframeofIMPCprojects,wealwaysuseC57Bl/6NpartnerstoobtainG1generationon100%B6background.

Evenintimesofprogrammablenucleasesandwidespreaduseof CRISPRs, the produced ESC resource stays awell-designedandveryusefultooltostudygeneexpressionasshowninFig.5andaschoicewhenconditionalallelesareinevitable.

Featured Article

Fig.4VelociGene’sKOMPDefinitiveNullAlleleDesign

References:

1. InternationalKnockoutMouseConsortium.Amouseforallreasons.Cell.2007;128:9–13.[PubMed:17218247]2. SkarnesWC,RosenB,WestAP,KoutsourakisM,BushellW,IyerV,MujicaAO,ThomasM,HarrowJ,CoxT,JacksonD,SeverinJ,BiggsP,FuJ,Nefedov

M,deJongPJ,StewartAF,BradleyA.(2011).Aconditionalknockoutresourceforthegenome-widestudyofmousegenefunction.Nature.474,337-342.

3. BirlingMC,DierichA,JacquotS,HéraultY,PavlovicG.(2012)Highly-efficient,fluorescent,locusdirectedcreandFlpOdeletermiceonapureC57BL/6Ngeneticbackground.Genesis.2012Jun;50(6):482-9.PMID:22121025

4. RyderE,DoeB, GleesonD, HoughtonR, DalviP, GrauE, HabibB, MiklejewskaE, NewmanS, SethiD, SinclairC, VyasS, Wardle-JonesH;SangerMouseGeneticsProject, BottomleyJ, BussellJ, GalliA, SalisburyJ, Ramirez-SolisR.(2014)RapidconversionofEUCOMM/KOMP-CSDallelesinmouseembryosusingacell-permeableCrerecombinase.TransgenicRes.2014Feb;23(1):177-85.

5. Valenzuela,D.M.,Murphy,A.J.,Frendewey,D.,Gale,N.W.,Economides,A.N.,Auerbach,W.,Poueymirou,W.T.,Adams,N.C.,Rojas,J.,Yasenchak,J.,etal.(2003).High-throughputengineeringofthemousegenomecoupledwithhigh-resolutionexpressionanalysis.NatBiotechnol21,652-659.

6. PettittSJ,LiangQ,RairdanXY,MoranJL,ProsserHM,BeierDR,LloydKC,BradleyA,SkarnesWC.(2009).AgoutiC57BL/6Nembryonicstemcellsformousegeneticresources.NatMethods.6,493-495.

7. PoueymirouWT,AuerbachW,FrendeweyD,HickeyJF,EscaravageJM,EsauL,DoréAT,StevensS,AdamsNC,DominguezMG,GaleNW,YancopoulosGD,DeChiaraTM,ValenzuelaDM.(2007).F0generationmicefullyderivedfromgene-targetedembryonicstemcellsallowingimmediatephenotypicanalyses.NatBiotechnol.Jan;25(1):91-9.PMID:17187059

Fig.5LacZstainingofthea)brain,b)liver,c)embryo,d)kidney,ande)testisincludingcaudaepididymis.

ba

c d e


Genome engineering has become popular again and morethan ever before in recent years with the availability ofprogrammable nucleases. Zinc finger nucleases (ZFNs) werethe first efficient programmable nucleases used for targetedgenomeengineering,asreviewedin(Urnovetal.2010).Here,a series of zinc fingers designed to bind a specific genomiclocusarefusedtoanucleasedomain(FokI)whichwhenpairedfacilitateaDNAdoublestrandbreak(DSB).Althougheffective,the assembly of ZFNs is time consuming and costly limitingtheirwideusebythescientificcommunity.Thischangedwhenthetranscriptionactivator-like(TAL)effectornuclease(TALEN)technology becamewidely available in 2011/ 2012 originallyfound in the plant-pathogenic bacterial genus Xanthomonas.Suddenly everyone was able to design and constructprogrammablenucleasesduetothesimplemodulardesignoftheTALEN.LikeZFNs,TALENconsistofaDNAbindingproteinfusedtoanucleasedomain(FokI)andfunctionasapair.UsingGoldenGateCloning(GGC)fortheassemblingofthesingleDNAbindingmodulesmadetheTALENarealsuccessandallowedawidespectrumofresearcherstouseTALENforgenomeediting.Thisway,TALENcouldbeassembledwithinaweekforaveryaffordableprice.However,whenmostofuswerestilltryingtofigureouttheBoch-Bogdanovecode(Bochetal.2009,Moscouand Bogdanove 2009) and setting up GGC reactions with 40differentplasmidsforoneTALENpair,anovelDNAeditingtoolwaslurkingaroundalready.TheClusteredregularlyinterspacedshort palindromic repeats (CRISPR)/CRISPR-associated (Cas)(CRISPR/Cas)systemsoriginallyfoundasadefencemechanisminarchaeaandbacteria includingtheStreptococcuspyogenesCas9(SpCas9)(Jineketal.2012),themostpopularCas-system

today.IncontrasttoZFNsandTALENs,CRISPR/Cas9iscomposedofaconstantproteinunit,thenucleasewhichisguidedtothetarget locus by a variable RNA component, the guide RNA(gRNA). The specificity of theCRISPR/Cas9 system is ensuredbyashort20nucleotide(nt)sequenceaspartofthegRNAplusa protospacer adjacentmotif (PAM)within the targetedDNAwhich is recognised by the Cas9 protein. The only part thatneedstobeadaptedforindividualtargetingisa20ntsequenceofthegRNA.Thus,makingtheCRISPR/Cas9systemveryflexibleand inexpensive for genome editing and enables even largegenomewide screens, asdemonstratedbyKoike-Yusaby theend of 2013 (Koike-Yusa et al. 2014) using a lentiviral librarycoveringtheentiremousegenome.Bynow,CRISPR/Cas9hasbeen adapted to a multitude of organisms and applications(Hsu,LanderandZhang2014,DoudnaandCharpentier2014) beyondgenomeeditingandbecameaglobalphenomenon.

Still,therearesomechallengesmostlyregardingthespecificity,restrictionsduetothePAMrequirementandsizeoftheCas9protein. Ifwe startwith the specificity, themain question is,ifashort20ntsequenceplusthePAMissufficienttoprovidethe specificity needed for precise and save genome editing?The short answer is yes, butwith limitations. Amultitude ofsophisticated techniques to precisely analyse off-targetinghave been developed including Guide-seq (Tsai et al. 2015),BreaksLabelingEnrichmentonStrepavidinandnextgenerationSequencing (BLESS) (Crosetto et al. 2013), LAM-PCR HTGTS(Frocketal.2015)andDigenome-seq(Kimetal.2015)toshedfurtherlightonoff-targeting(alsosummarizedin(Koo,LeeandKim2015)),revealingthatthereareindeedoff-targeteffectsandsomeofthemareevendifficulttopredictbycurrentsoftwaretools.Whilethesefindingsreflectresultsfromcellcultures,thesituation in targetedmiceseemstotallydifferentwithalmostzerodetectableofftargetmutations(Iyeretal.2015).

Already early on the mechanism how CRISPR/Cas9 bindsto its targetDNAand the toleranceofmismatches has beeninvestigated (Fu et al. 2013, Hsu et al. 2013). Thanks to thisfindings our design of gRNAs significantly improved to avoidundesired effects in off-target sites. As a rule of thumb, theactivityoftheCRISPRdependsonthenumberofmismatchesand their position. Here, the mismatches within the seedsequenceofthegRNA(thesequenceproximaltothePAM)haveastrongereffectontheactivitythanatthe5’endofthegRNA.With this in mind, clever selection of gRNAs avoids alreadynegativesideeffects.ButalsomodificationsofthegRNAscanimprove the targeting specificity. The intentional addition oftwomismatchesatthe5’endofthegRNA(e.g.startingwithGG)(Kimetal.2015,Choetal.2014)ortheuseoftruncatedgRNAs(Fuetal.2014)wereshowntoreduceoff-targeteffectssignificantly. But improvements don’t stop here. The use ofCas9proteininsteadofplasmidsshortenthedurationofactivenucleasecomplexeswithinthecells,thusfurtherreducingthechance of accumulating off-target effects in cells (Kim et al.2014).

Bjoern SchusterTargeting Unit

An Overview of programmable nuclease technology

Featured Review

Figure 1. adapted from Doudna& Charpentier(2014) Science,showing the potential of future development of the CRISPR/Cas9 system. Only two years later the vision became realitywithprogrammablenucleasesbeingpresent inmultipleareasinbiomedicineandbiotechnologywithanstillexpandingfieldofapplications.

ProgrammablenucleaseZNF-TALEN-CRISPR

HumanGeneTherapy Animaldisease

models

Agriculture:cropsandanimals

SyntheticbiologyRNA

targeting

Viralgenedisruption

Ecologicalvectorcontrol

Drugtargetscreens


Othersolutions,tofurthereliminateoff-targetingincludeCas9Nickases were the Cas9 facilitates a nick in a single strandinsteadofaDSB(Malietal.2013,Ranetal.2013)orlikewisetoZFNsorTALEN,deadCas9(dCas9)fusedtoFokI(Tsaietal.2014)canbeused.SinceherearealwaystwogRNAsrequiredinsteadofone,theefficiencyofthenucleasesmightbelower.

ImproveddesignofgRNAsandexperimental setupsmightbesufficientformostapplicationsinbiologicalresearch,butitstilldoesn’tfulfilrequirementsforprecisiongenetherapyinhumanwereahighlevelofspecificityisrequiredandtheimprovementinspecificityisthereforeinfocusofcurrentresearch.Recentlytwoindependent groups developed structure-guided engineeredSpCas9variantswithreducedoff-targeting,yetstrongon-targetactivitybydestabilisingthebindingoftheCRISPR/Cas9complextoDNA.Thiswasachievedbyeitherweakeningthebindingtothedisplaced,non-targetedDNAstrandwithenhancedSpCas9(eSpCas9)(Slaymakeretal.2016)orthetargetedstrandusinghighfidelity SpCas9-HF1 (Kleinstiver et al. 2016a) resulting innucleaseswithvirtuallynooff-targeteffects.

InassimilarapproachSpCas9wasalteredtobindtoalternativePAMsequences.WhilethePAMsequenceprovidesadditionalspecificityoftheCRISPR/Cas9systemitcanontheotherhandleadtocertain limitations.ThenaturalSpCas9PAMsequenceNGG allows targeting of virtually any gene in the genomefor gene depletion. However, the PAM requirement mightbe problematic for precise genome editing in GC low loci orwhenallelespecifictargetingisrequired.Toaddressthisissue,engineered Cas9 proteins were developed using alternativePAM sequences (Kleinstiver et al. 2015b, Kleinstiver et al.2015a)broadeningthespectrumofgenomictargetloci.WhilethisstrategyisstillbasedontheSpCas9tooptimizethesystem,alternativenucleasehavebeenexplored.

AlternativestotheCRISPR/Cas9system

Hitherto,SpCas9isthemostwidelyusedprogrammablenuclease.However,therearesomeinterestingalternativeswhichdeserveourattention,includingtheSaCas9fromStaphylococcusaureus(Ranetal.2015)andtheCas9-likeproteinCpf1(Zetscheetal.2015).Bothprogrammablenucleasesperforminacomparable

waytoSpCas9,buttheyhaveonemajoradvantage.Theyaresignificantly smaller in size which is favourable especially forapplicationswhereviralvectorsystemshavetobeused.TheyalsoutilizealternativePAMsequencescomparedtoSpCas9andthereforecomplementournucleasetoolboxfurtherbroadeningthespectrumofgenomictargetlociforprecisegenomeediting.Cpf1hasalreadybeenproventobesuitableformousemodelgeneration(Kimetal.2016b)andtherearefirstindicationsthatCpf1mightpossessevenhigher specificity compared towild-typeSpCas9(Kimetal.2016a,Kleinstiveretal.2016b).TogetherwiththeevensimplerdesignoftheCpf1gRNAandthedifferentcuttingmode, resulting inDSBwith short over-hangs insteadofbluntendcausedbyCas9,Cpf1representsaveryattractivealternativetoSpCas9.

Another putative alternative to SpCas9 could be Argonaute,anendonucleaseoriginating fromNatronobacterium gregoryi Argonaute (NgAgo) (Gaoetal.2016). Incontrast to theaforementionednucleases,NgAgoisassDNA-guidednucleasewitha 24nt gDNA insteadof a gRNAwithout theneedof a PAMsequence. However, there are currently some controversiesaboutthereproducibilityoftheinitialreports.

As genome editing tools ZFNs, TALEN and CRISPR facilitate adoublestrandbreakinDNA,that’sit.

TheDSBisrepairedbytheDNArepairmachineriesofthecellbytwomajorpathways, thenon-homologousend joining (NHEJ)orhomology-directedrecombination(HDR).WhileNHEJiserrorproneitisthemostefficientwaytointroducesmallinsertionsor deletionswithin gene coding regions to cause frame shiftmutationsleadingtogenedepletion.TheCRISPRCas9systemmakes it easy to evenmultiplex gene targeting by supplyingmultiple gRNAs at the same time (Cong et al. 2013). Whilethis is ideal for studying the function of the gene products,the editing is not precise and somehow unpredictable. Incontrast, HDR can be employed to specifically modify thegenomebysimultaneouslyprovidingthenucleaseandaDNArepair template to the cells. However, HDR is less efficientcomparedtoNHEJandiscurrentlyoneofthemajorchallengesto solve.Attempts to improveHDRevents include theuseof

Featured Review

Targeted insertionsOne nuclease + repair template

Programmable nucleaseZNF – TALEN - CRISPR

Indel mutationsOne nuclease

Inversions and DeletionTwo nucleases

Targeted insertionsOne nuclease + repair template

NHEJ/MMEJ HDR NHEJ/MMEJ

Figure 2. Programmable nucleases in genome editingapplications. As nucleases, ZFN, TALEN and CRISPRmediateaDNAdouble strandbreak (DBS) at target loci. CellularrepairmechanismsfixtheDSBbyerrorprone end joining either non-homologous (NHEJ) ormicro-homologymediated(MMEJ)idealforgenedisruption by introducing out of frame mutations. Incontrast,homologydirectedrepair(HDR)inpresence of a donor template, ssDNAor dsDNA, allowsprecisiongenomemodifications.Applyingtwonucleasessimultaneously,genomicregionscanbedeletedorinvertedbyNHEJ/MMEJ.


Section TitleFeatured Review

inhibitorsofNHEJortopromoteHDRrepair(Chuetal.2015).ButalsothedesignoftheDNAdonorconstructshaschangeddramaticallywithCRISPR/Cas9from‘classical’targetingvectors,used in conjunctionwithprogrammablenucleases containinghomology arms of 700-1000 bp length, to homology armindependent targeting constructs seem towork sufficient forbiologicalapplications(Yoshimietal.2016,Suzukietal.2016).Thus,simplifyingnotonlythedesignoftargetingconstructsbutalso the downstream genotyping significantly. However, thedestructivenatureof thenuclease remainsstill stronger than

thepreciserepairoftheDBS.Seeingtheprogressthathasbeenmadeinrecentyearsandthespeedofimprovementsineveryaspectofgenomeeditingitseemsjustamatteroftimetillthelastobstaclesare solvedpaving theway for saveandprecisegenomicmodificationsinresearchandhumantherapy.

References:

1. Boch,J.,H.Scholze,S.Schornack,A.Landgraf,S.Hahn,S.Kay,T.Lahaye,A.Nickstadt&U.Bonas(2009)BreakingthecodeofDNAbindingspecificityofTAL-typeIIIeffectors.Science,326,1509-12.

2. Cho,S.W.,S.Kim,Y.Kim,J.Kweon,H.S.Kim,S.Bae&J.S.Kim(2014)Analysisofoff-targeteffectsofCRISPR/Cas-derivedRNA-guidedendonucleas-esandnickases.GenomeRes,24,132-41.

3. Chu,V.T.,T.Weber,B.Wefers,W.Wurst,S.Sander,K.Rajewsky&R.Kuhn(2015)Increasingtheefficiencyofhomology-directedrepairforCRIS-PR-Cas9-inducedprecisegeneeditinginmammaliancells.NatBiotechnol,33,543-8.

4. Cong,L.,F.A.Ran,D.Cox,S.Lin,R.Barretto,N.Habib,P.D.Hsu,X.Wu,W.Jiang,L.A.Marraffini&F.Zhang(2013)MultiplexgenomeengineeringusingCRISPR/Cassystems.Science,339,819-23.

5. Crosetto,N.,A.Mitra,M.J.Silva,M.Bienko,N.Dojer,Q.Wang,E.Karaca,R.Chiarle,M.Skrzypczak,K.Ginalski,P.Pasero,M.Rowicka&I.Dikic(2013)Nucleotide-resolutionDNAdouble-strandbreakmappingbynext-generationsequencing.NatMethods,10,361-5.

6. Doudna,J.A.&E.Charpentier(2014)Genomeediting.ThenewfrontierofgenomeengineeringwithCRISPR-Cas9.Science,346,1258096.7. Frock,R.L.,J.Hu,R.M.Meyers,Y.J.Ho,E.Kii&F.W.Alt(2015)Genome-widedetectionofDNAdouble-strandedbreaksinducedbyengineered

nucleases.NatBiotechnol,33,179-86.8. Fu,Y.,J.A.Foden,C.Khayter,M.L.Maeder,D.Reyon,J.K.Joung&J.D.Sander(2013)High-frequencyoff-targetmutagenesisinducedbyCRISPR-Cas

nucleasesinhumancells.NatBiotechnol,31,822-6.9. Fu,Y.,J.D.Sander,D.Reyon,V.M.Cascio&J.K.Joung(2014)ImprovingCRISPR-CasnucleasespecificityusingtruncatedguideRNAs.NatBiotechnol,

32,279-84.10. Gao,F.,X.Z.Shen,F.Jiang,Y.Wu&C.Han(2016)DNA-guidedgenomeeditingusingtheNatronobacteriumgregoryiArgonaute.NatBiotechnol,34,

768-73.11. Hsu,P.D.,E.S.Lander&F.Zhang(2014)DevelopmentandapplicationsofCRISPR-Cas9forgenomeengineering.Cell,157,1262-78.12. Hsu,P.D.,D.A.Scott,J.A.Weinstein,F.A.Ran,S.Konermann,V.Agarwala,Y.Li,E.J.Fine,X.Wu,O.Shalem,T.J.Cradick,L.A.Marraffini,G.Bao&F.

Zhang(2013)DNAtargetingspecificityofRNA-guidedCas9nucleases.NatBiotechnol,31,827-32.13. Iyer,V.,B.Shen,W.Zhang,A.Hodgkins,T.Keane,X.Huang&W.C.Skarnes(2015)Off-targetmutationsarerareinCas9-modifiedmice.NatMeth-

ods,12,479.14. Jinek,M.,K.Chylinski,I.Fonfara,M.Hauer,J.A.Doudna&E.Charpentier(2012)Aprogrammabledual-RNA-guidedDNAendonucleaseinadaptive

bacterialimmunity.Science,337,816-21.15. Kim,D.,S.Bae,J.Park,E.Kim,S.Kim,H.R.Yu,J.Hwang,J.I.Kim&J.S.Kim(2015)Digenome-seq:genome-wideprofilingofCRISPR-Cas9off-target

effectsinhumancells.NatMethods,12,237-43,1pfollowing243.16. Kim,D.,J.Kim,J.K.Hur,K.W.Been,S.H.Yoon&J.S.Kim(2016a)Genome-wideanalysisrevealsspecificitiesofCpf1endonucleasesinhumancells.

NatBiotechnol,34,863-8.17. Kim,S.,D.Kim,S.W.Cho,J.Kim&J.S.Kim(2014)HighlyefficientRNA-guidedgenomeeditinginhumancellsviadeliveryofpurifiedCas9ribonu-

cleoproteins.GenomeRes,24,1012-9.18. Kim,Y.,S.A.Cheong,J.G.Lee,S.W.Lee,M.S.Lee,I.J.Baek&Y.H.Sung(2016b)GenerationofknockoutmicebyCpf1-mediatedgenetargeting.Nat

Biotechnol,34,808-10.19. Kleinstiver,B.P.,V.Pattanayak,M.S.Prew,S.Q.Tsai,N.T.Nguyen,Z.Zheng&J.K.Joung(2016a)High-fidelityCRISPR-Cas9nucleaseswithnodetect-

ablegenome-wideoff-targeteffects.Nature,529,490-5.20. Kleinstiver,B.P.,M.S.Prew,S.Q.Tsai,N.T.Nguyen,V.V.Topkar,Z.Zheng&J.K.Joung(2015a)BroadeningthetargetingrangeofStaphylococcus

aureusCRISPR-Cas9bymodifyingPAMrecognition.NatBiotechnol,33,1293-1298.21. Kleinstiver,B.P.,M.S.Prew,S.Q.Tsai,V.V.Topkar,N.T.Nguyen,Z.Zheng,A.P.Gonzales,Z.Li,R.T.Peterson,J.R.Yeh,M.J.Aryee&J.K.Joung(2015b)

EngineeredCRISPR-Cas9nucleaseswithalteredPAMspecificities.Nature,523,481-5.22. Kleinstiver,B.P.,S.Q.Tsai,M.S.Prew,N.T.Nguyen,M.M.Welch,J.M.Lopez,Z.R.McCaw,M.J.Aryee&J.K.Joung(2016b)Genome-widespecificities

ofCRISPR-CasCpf1nucleasesinhumancells.NatBiotechnol,34,869-74.23. Koike-Yusa,H.,Y.Li,E.P.Tan,C.Velasco-HerreraMdel&K.Yusa(2014)Genome-widerecessivegeneticscreeninginmammaliancellswithalentiviral

CRISPR-guideRNAlibrary.NatBiotechnol,32,267-73.24. Koo,T.,J.Lee&J.S.Kim(2015)MeasuringandReducingOff-TargetActivitiesofProgrammableNucleasesIncludingCRISPR-Cas9.MolCells,38,475-81.25. Mali, P., J.Aach, P.B. Stranges, K.M. Esvelt,M.Moosburner, S. Kosuri, L. Yang&G.M.Church (2013)CAS9 transcriptional activators for target

specificityscreeningandpairednickasesforcooperativegenomeengineering.NatBiotechnol,31,833-8.26. Moscou,M.J.&A.J.Bogdanove(2009)AsimpleciphergovernsDNArecognitionbyTALeffectors.Science,326,1501.27. Ran,F.A.,L.Cong,W.X.Yan,D.A.Scott,J.S.Gootenberg,A.J.Kriz,B.Zetsche,O.Shalem,X.Wu,K.S.Makarova,E.V.Koonin,P.A.Sharp&F.Zhang

(2015)InvivogenomeeditingusingStaphylococcusaureusCas9.Nature,520,186-91.28. Ran,F.A.,P.D.Hsu,C.Y.Lin,J.S.Gootenberg,S.Konermann,A.E.Trevino,D.A.Scott,A.Inoue,S.Matoba,Y.Zhang&F.Zhang(2013)Doublenicking

byRNA-guidedCRISPRCas9forenhancedgenomeeditingspecificity.Cell,154,1380-9.29. Slaymaker,I.M.,L.Gao,B.Zetsche,D.A.Scott,W.X.Yan&F.Zhang(2016)RationallyengineeredCas9nucleaseswithimprovedspecificity.Science,

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30. Suzuki,K.,Y.Tsunekawa,R.Hernandez-Benitez,J.Wu,J.Zhu,E.J.Kim,F.Hatanaka,M.Yamamoto,T.Araoka,Z.Li,M.Kurita,T.Hishida,M.Li,E.Aizawa,S.Guo,S.Chen,A.Goebl,R.D.Soligalla,J.Qu,T.Jiang,X.Fu,M.Jafari,C.R.Esteban,W.T.Berggren,J.Lajara,E.Nunez-Delicado,P.Guillen,J.M.Campistol,F.Matsuzaki,G.H.Liu,P.Magistretti,K.Zhang,E.M.Callaway,K.Zhang&J.C.Belmonte(2016)InvivogenomeeditingviaCRISPR/Cas9mediatedhomology-independenttargetedintegration.Nature.

31. Tsai,S.Q.,N.Wyvekens,C.Khayter,J.A.Foden,V.Thapar,D.Reyon,M.J.Goodwin,M.J.Aryee&J.K.Joung(2014)DimericCRISPRRNA-guidedFokInucleasesforhighlyspecificgenomeediting.NatBiotechnol,32,569-76.

32. Tsai,S.Q.,Z.Zheng,N.T.Nguyen,M.Liebers,V.V.Topkar,V.Thapar,N.Wyvekens,C.Khayter,A.J.Iafrate,L.P.Le,M.J.Aryee&J.K.Joung(2015)GUIDE-seqenablesgenome-wideprofilingofoff-targetcleavagebyCRISPR-Casnucleases.NatBiotechnol,33,187-97.

33. Urnov,F.D.,E.J.Rebar,M.C.Holmes,H.S.Zhang&P.D.Gregory(2010)Genomeeditingwithengineeredzincfingernucleases.NatRevGenet,11,636-46.

34. Yoshimi,K.,Y.Kunihiro,T.Kaneko,H.Nagahora,B.Voigt&T.Mashimo(2016)ssODN-mediatedknock-inwithCRISPR-Casforlargegenomicregionsinzygotes.NatCommun,7,10431.

35. Zetsche,B.,J.S.Gootenberg,O.O.Abudayyeh,I.M.Slaymaker,K.S.Makarova,P.Essletzbichler,S.E.Volz,J.Joung,J.vanderOost,A.Regev,E.V.Koonin&F.Zhang(2015)Cpf1isasingleRNA-guidedendonucleaseofaclass2CRISPR-Cassystem.Cell,163,759-71.

Metabolism, from cells to mouse

Spring 2017Weare glad to announceourmetabolicworkshop “Metabolism from cells tomouse”. Thisworkshopwill be co-organizedwith Agilent Technologies. During this workshopwewill demonstrate Seahorse technology, Q-TOF andindirectcalorimetryall inoneexperimentalmodel.WewillshowhighthroughputmethodsasSeahorsewithmoredetailedmetabolitescreenbyQ-TOFandtranslationofcellcultureresultsitintolivingmousemodelusingindirectcalorimetry.Pleaseseemoredetailsonourwebsitephenogenomics.cz.



Researchintometabolicdisordersandtheirassociateddiseasesisvitalforbothunderstandingandtreatment.Althoughdiseasessuchasheartdisease,strokesandcancersarecloselyaffectedbymetabolicdisorders,diabetesmellitusandobesityaremajormetabolic diseases affecting our society. The incidences ofbothdiabetes(particularlyobesity-relatedtype2diabetes)andobesityinbothdevelopedanddevelopingsocietieshasformedthe basis of research into understanding the onset of thesedisordersandalsofindingbettertreatments1.

Diabetesmellitus,definedbyaphysicalorfunctionallossofβcells, is characterizedby thepersistenceofhigh,unregulatedbloodglucose levels. Leftuntreated, thisdiseasecan lead toserious damage to the heart, blood vessels, eyes, kidneys,andnervesandcomplicationscanleadtoheartattack,stroke,blindness, kidney failure and lower limb amputation. Withmillionsofpeoplecurrentlylivingwiththedisease,WHOhas,aspartof the2030Agenda forSustainableDevelopment, settheambitioustargettoreduceprematuremortalityfromnon-communicablediseases–includingdiabetesby20302.

Obesity,isdefinedbytheabnormalorexcessiveaccumulationoffatthatmayimpairhealth.In2008,morethan1.4billionadultswere diagnosed as overweight and more than half a billionwereobese(WHO).Ofmoreconcern, is therise inchildhoodobesity,globally,42millionpreschoolchildrenwereoverweightin2013(WHO).Theroleofobesityinotherdiseaseshasbeenwell characterised, with 44% of diabetes, 23% of ischaemicheartdiseaseand7–41%ofcertaincancersareattributabletooverweightandobesity3.

Whilst research using in vitro models forms a powerful toolforansweringmanyquestions,theuseofinvivomodelsallowinvestigationintothecomplexnetworkofsystemsaffectedbybothdisorders. Inadditiontothat,useoftransgenicmodels,wherespecificgenesareeitherupordownregulatedfurtherstrengthentheroleofinvivostudiesintothesedisorders.

Themetabolismunit at theCzechCentre for Phenogenomicsaimstosupporttheseresearchpursuitsintometabolicdiseasesusingtransgenic,invivomouseandratmodels.AsamemberoftheInternationalMousePhenotypeConsortium(IMPC),CCPisinvolvedinthehigh-throughputphenotypingofknockoutmicetodiscoverthefunctionofeverygene.Aspartofthissystematicphenotyping pipeline, themetabolism unit conducts indirectcalorimetrytestsandtheintraperitonealglucosetolerancetest(IPGTT).Usingthesetworobusttests,theunitisabletoidentifysubtlemetabolicchangesinunrestrainedanimals.

Our standardised screen has been developed as part of theInternational Mouse Phenotyping Resource of StandardisedScreens International Mouse Phenotyping Resource ofStandardisedScreens(IMPReSS).Thesetestscanalsobeusefulasprimaryscreensforresearchersandgroupleadersinterestedin understanding the metabolic function of their specificresearchmodels.

Energy expenditure

Indirect calorimetry is a non-invasive method where oxygenconsumptionandcarbondioxideproductionaremeasuredoveragivenperiodoftimeinahomecageenvironment.Thepurposeofthesemeasurementsistoprovideinformationaboutenergymetabolismand todetect abnormalitiesof carbohydrate andlipidmetabolism inrodents.Sinceoxygenandcarbondioxideare measured simultaneously, the respiratory exchange ratio(RER)canbecalculated;Fig.1).Asactivity,foodandwaterintakearealsomonitored,totalenergyexpenditurecanbeevaluated4.

Nicole ChambersMetabolism Unit

Metabolism Unit

Featured Service

Figure 1. Representive trace depicting alterations in RER(RespiratioryExchangeRatio) as seenusingourPhenomasterIndirectcalorimetrysystem.

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Figure 2.Exampleofaglucosetolerancetestindicatingimpariedglucoseclearenceinthegroupdepictedinred.


Intraperitoneal glucose tolerance test (IPGTT)

This test is used for detection of abnormalities in glucosemetabolism.Inhumanmedicalpracticethistestisperformedtodiagnosediabetesandmetabolicsyndrome.Thetestissimilarformiceandhumans;itmeasurestheclearanceofaglucoseloadfromthebodyatdifferenttimepoints.Micearefastedfor16h,basalglucoselevelismeasuredpriortoglucoseintraperitonealinjectionandglucoselevelsaremeasuredduringthefollowinghours(Fig.2).

Insulin blood level

Insulin isapeptidehormone,which isan importantregulatorof glucosehomeostasis. The levelof insulin inmouseplasmais detected during terminal bleeding. Together with theintraperitoneal glucose tolerance test, an abnormal level ofinsulinisindicatorofdiabeticormetabolismrelatedphenotype.

Rat models of metabolic disorders

The metabolic unit is striving for both the standard andcustomised services to be available for both mouse and ratmodels. We believe that offering services such as Indirectcalorimetryandpair-feeingstudiesforratmodelswillprovetobe valuable to researchers. When this is combinedwith theabilitytogeneratetransgenicratmodels,themetabolismunitcanaidetheinvestigationtheroleofaspecificgene(ormultiplegenesinaparticularpathway)invariousmetabolicdisorders.Formoreinformationonusingratmodelsortodiscussparticularexperimentsyoucancontactusatccp@phenogenomics.cz.

In conclusion, the metabolism unit at the Czech Centre forPhenogenomics ready to aide research projects investigatingthemechanismsofvariousmetabolicdisorders.Ourstandardcustomisedtests(Table1),makeuseofstateofthearttechnicalequipmentsuchasthePhenomasterSystemfromTSEsystemsandcanbeconductedonbothmouseandrat5models.Whilstour standard tests allow a general overview of metabolicabnormalities, the customised tests focus on more in depthinvestigationofmetabolicdiseasemodels andutalisethelatesttechnology from complementary units such as Bioimaging,ClinicalChemistryandhistopathology.Alltestsdescribedinthisarticlearecurrentlyavailableandcanbeadaptedtothespecificrequirementsofindividualresearchprojects.

For more information about our services visit our websitehttp://www.phenogenomics.cz/phenotyping/.

Figure 3:Customisedservices:A)Bodyscandepictingfat,leanmassandbonecourtesyofthebodyimagingunit.B)Insulinstainofthepancreaticislet,courtesyofthehistopathologyunit.C)Phenomasterindirectcalorimetrycageswithpairedfeedingcapability.

A B C

Featured Service

References:

1. BoweJE.etal.(2014)MetabolicPhenotypingguidelines:Assessingglucosehomeostasisinrodentmodels.JournalofEndocrinology.222:3.G13-G252. Globalreportondiabetes.WorldHealthOrganization(2016).3. 10FactsonObesity.WorldHealthOrganization(2014).4. TschopMH,etal.(2012)Aguidetoanalysisofmoueenergymetabolism.NatureMethods.9(1),57-63.5. KingAJF,etal.(2012)Theuseofanimalmodelsindiabetesresearch.BrJPharmacol.166(3).877-894.

Standard Services:• EnergyExpenditure• IPGTT• InsulinToleranceTest

Customised Services:• Pair-feedingstudies• Highfatfedrodentmodel• Diabetesinducedinrodentmodels• Bodycomposition

Table 1. Overview of the services offered by the Metabolismunit.


Introdcution

Alterations of the lung function, the ability of the lungs toefficiently exchange gasses, is a key factor of pulmonarydiseasesandthereforealsoacriticalparametertobemeasuredinanimalmodelsofthesediseases.

There are 2 types of lung function measurements. In thefirst and most used type, mechanical parameters of thelung are measured; these include resistance to the airflowand compliance (elasticity related parameter) of the lungs. Theseparametersmainlyinfluencetheairflowandtherenewalofairwhichcanbeachieved in the lung.The second typeofmeasurementsismeasuringthegas-exchangeinsidethelungs.It is done by injecting a known gas mixture in the lung andmeasuring the composition of the exhaled gas. As the lattermethodisonlyrarelyused,itwillnotbediscussedinthisarticle.

The early years: Indirect measurements on explants

Many patients have episodes of reversible acute respiratorydistress, caused by increased sensitivity of the airways tonon-specific stimuli like cold-air or dusty environments. Thisincreasedsensitivityiscalled‘airwayhyperreactivity’(AHR).OneofthemainmechanismscausingAHR,istheexaggeratedcontractionofthesmoothmusclelayersurroundingtheairway.In many lung diseases, this layer also shows hypertrophy,makingitcontractevenharder.

This contractile force is what was measured in the firstmethods to assess AHR. Rings of the trachea or pieces ofthe smooth muscle layer surrounding the trachea wereisolatedandattached toa force-transducer inanorganbath. Explantswerethenexposedtoacetylcholineormethacholine(a non-specific bronchoconstrictive agent) and contractionforcewasmeasured.

The major drawback of the indirect methods is thatmeasurements are done in small parts of the pulmonarysystem and might thus not be representative for the wholesystem.Additionally the lung function isnotonlydeterminedbythecontractileforceofsmoothmusclelayersurroundingtheairways.

Measurements of lung function in ventilated animals

Airway pressure-time index

The development of ventilators for small animals led to thepossibility to measure lung mechanics parameters in livinganaesthetized animals and thus measure the completepulmonary system. The first method that was developedwas the measurement of the ‘airway pressure time index’,abbreviatedasAPTI.Forthisanimalsweretracheotomizedandattachedtoasmallanimalventilatorwithapressuretransducerattachedattheairwayopening.TheAPTIwasthencalculatedastheareaunderthecurveofpressureversustimecurveforone

exhalation.Usuallymicealsohadan i.v. line inserted in theirjugular vein to administer methacholine (or acethylcholine)and thus record dose response curves and determine AHR. This method was a huge step forwards compared tomeasurements on explants, but itwas still very rudimentary,asittellsyouthereissomerestrictionintheflow,butdoesn’tinformaboutwhatiscausingthisrestriction.Thesecausescanbemultiple,oneofthemtheresistancetotheairflowcausedbythecontractionoftheairwayswasalreadydiscussed.Thesecondmaincauseofsuboptimalairflowisthelossofelasticityofthelungs,which is typical indiseases likepulmonaryemphysemaandpulmonaryfibrosis.Duetothislossofelasticitythelungscannotinflatethemselveswithairoptimallyandcannotdeflateas the elasticity of the lungs is the main driving force fordeflation,especiallyinsmallanimals.

Plethysmography in ventilated animals: Resistance and compliance

Todeterminetheresistanceandtheelasticityofthelungsoneneedtodetermine3parameters,i.e.theflowofair,thevolumeofairpumpedinthelungsandthepressurethisvolumeofaircauses in the thorax.With theAPTImeasurementswe couldalreadydeterminethepressure,withthepressuretransducerat the airwayopening, and the volume,which is determinedby the settings of the ventilator. So amethod needed to befound to determine the flow, but this is challenging in miceandother small animals as the flow is very small due to thesmall size of the lungs, whichmakes directmeasurement oftheflowchallenging.A solutionwas foundwith theuseof aplethysmographychamber.Thesechambersarerigidboxes inwhichpressureismeasured.Thechangeinpressurecausedbytheventilationoftheanimalwhichisplacedintheboxcorrelateswellwith theairflowandcan thusbeused todetermine theairflowindirectly.Anebulizercanbeattachedinlinewiththe

Benoit PiavauxLung Function Unit

Lung function measurement in laboratory animals: An historical overview

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Figure 1: Typical set-up for advanced lung mechanicsmeasurementsbaseduponplethysmography.


ventilatortobeabletoadministerbroncho-constrictiveagentsandthusdetermineAHR.

Somevariationsofthismethodhavebeendeveloped,mainlytoapplyittorestrainedbutunanaesthetizedanimals.Theyusuallyuse 2 chamber plethysmograph set-upswhere thebody is inanother plethysmograph chamber than the nose. The airwayresistance is derived from the difference between the flowof the nose-chamber (mouse is exclusive nose-breather) andthebodychamber. Itdoesnotallowthemeasurementofthelung complianceas there isnomeasurementof thepressurein the lungs.Therearealsohead-outplethysmographswhichonly measure the thoracic flow in unanaesthetized animals.Thislattermethoddoesn’tallowforthecalculationoftruelungmechanicsparameters.

Plethysmography in ventilated animals: advanced methods

Based upon the same principles of the resistance andcompliancemeasurementsmoreadvancedmeasurementsweredeveloped.Theflow,pressureandvolumearestilldeterminedthesameway,butmeasurementslikebreathholdsandforcedexpiratory and inspiratory maneuvers were added (Fig. 1). Despitethefactthatthesemachinesareabletogeneratemanymore parameters and especially parameters which are alsomeasured intheclinic inhumanpatients, theyneverbecamevery popular. The main reason for this is that at the timethese set-ups where launched the unrestrained whole bodyplethysmographywasverypopular,asitwasverysimpletouseandmicedidn’tneedtobeanesthetizedandtracheotomized.

Advanced methods without plethysmography

The latest generation of machines for lung mechanicsmeasurementsdoesnotuseplethysmography.Insteadofthat,theyusemathematicalmodellingofthepressurecurvesfroma transducer placed at the ventilator piston and a pressuretransducer placed on the expiratory line. As this method ismore robust and the results more reproducible than withplethysmographybasedset-ups,itquicklybecamethe‘goldenstandard’ for lung function measurements in laboratoryanimals. Additionally the machines allow for perturbationsat different frequencies, by which one can determine if thechangesinlungfunctionareduetochangesintheairwaysorthelungparenchyma(Fig.2).

Unrestrained whole body plethysmography does not measure lung-function

An overview of the devices for lung-function measurementwouldnotbe completewithoutmentioning theunrestrainedwholebodyplethysmography.Ithasbeenthemostusedmethodfor lungfunctionassessmentformanyyears. Intheseset-upsthe mouse is freely moving in a small plethysmography boxandrespiratorycurvesaremeasured.Thesemachinesallowedhighthroughputscreeningas1set-upcouldhandle12boxes.Mice can be challenged via methacholine or acetylcholineaersoldeliveredbyanebulizer.Fromthemeasuredrespiratorycurves a parameter called: ‘enhanced pause’, abbreviated:‘penh’ is calculated. Unfortunately it was shown by manyauthorsthatthepenhdoesnotcorrelatetoanymeasurementof lung mechanics parameters. Therefore the measurementsmadewithunrestrainedwholebodyplethysmographyarenowconsideredunreliable.Somelabsstilluseitasahighthroughputpre-screeningmethodandconfirmtheirfindingslaterbymorereliablemethods.Asthecorrelationisbadorevencompletelyabsentbetweentheunrestrainedplethysmographyandothermore direct methods for lung-function measurements, thevalue,ifany,ofthispre-sreeningremainstobedetermined.

Conclusion

Since the start of lung function measurements there havebeenmanymethodsdevelopedtoquantifydeviations in lungfunctionparameters,fromveryartificialorganbathsonexplantsofthetracheatotheveryelaboratemachineswhichmeasurelung function nowadays andwhich offer an expanding rangeofoptionsandmodules.AtCCPwehavechosenforthelatestgenerationmachines,whichdonotrelyonplethysmographyformeasurementsonasinglesubject.Wehaveaplethysmographybased multiple subject extension for these machines whichallowustomeasureresistanceandcompliance,withorwithoutmethacholine challenge, in 8 animals simultaneously for high-throughputstudies.

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Figure2:LatestgenerationlungfunctionequipmentasavailableatCCP.Themachineinthebackistheset-upasitwouldbeusedforassessmentoflungfunctioninasingleanimal.Themoduleinthefront isthehigh-throughputresistanceandcomplianceplethysmographymodulefor4mice.(PicturefromScireq,usedwithpermission).

Further Reading:

1. BerndtA.,etal.(2011)Comparisonofunrestrainedplethysmographyandforcedoscillationforidentifyinggeneticvariabilityofairwayresponsive-nessininbredmice.PhysiolGenomics.43(1):1–11.

2. IrvinCG,BatesJH.(2003)Measuringthelungfunctioninthemouse:thechallengeofsize.RespirRes.4(1):1.3. ZhangQ.,etal.(2009)Doesunrestrainedsingle-chamberplethysmographyprovideavalidassessmentofairwayresponsivenessinallergicBALB/c

mice?RespiratoryResearch.10(1):61.


Idovisitmanyanimalfacilitiesworldwideforseveralreasons;to check their ability to get the necessary legal licences toworkwith laboratory animals, to consult on how to improvetheirmaterialandpersonalflowor justasa friendlyvisit.Allfacility managers, including myself, have the same way howtopresentthequalityoftheirsites.It isusuallyaguidedtourthroughtheanimalfacilitybuildingwhichincludesdescriptionofhousingsystem,decontaminationtechnology,drinkingwatertreatment, cage washing technology and sterilization, HVACtechnologyandmaterialandpersonalflowwithhugeamountofinformationwhythistechnologyisbetterthanothers.Suchinformationisveryusefultogetanideaaboutdefaultqualityof the facility, but although youwill have thebest cages andhousing technology,youcanstilldealwithanimalhealthandwelfareproblems,whichcaninfluencethevalidityofscientificdataobtainedfromanimals.Amorevaluablequalitativefactoris that intangible thing hidden behind the scene, which isusuallynotvisibleduringashortsitevisit.Wearetalkingaboutthe “soft” part of husbandry. Husbandry is described in theAssistantLaboratoryAnimalTechnicianTrainingManualas :“Thepracticeofprovidingappropriatesupportforthephysicaland psychological well-being of the animals. ” This includesa lotof things likedifferentmethodsofanimal identification,type of the bedding, diet and its storage and distribution,differenttypesofwatering,environmentalcontrolandanimalmanagementsystem.Properselectionofallmaterialsandit’srelation to species, work flow, personnel flow, material flowand scientific needs specific to your animal facility is a veryimportant factor which must be considered well by facilitymanagersandwritten inSpecificoperationalprocedures.Thekeypartofsuccessareanimalfacilityemployees.Eachmemberoftheteamisancharacterwithdifferentneeds,motivationandideaswhichgivesonmanagerslotofresponsibilitytokeeptheminterestedintheLabanimalfieldtoincreasetheirprofessionalvalue, attitude, responsibility level andmore - to change thecultureofcare.

Here at the Czech Centre for Phenogenomics we haveestablisheda stateof theartanimal facility. This facilitywasbuiltwiththesolepurposeofservingtheresearchcommunityinboththeCRandwiderinternationalcommunity.Theentireanimal facility is separated into two locations with themainbreedingandproductionlocatedinthebrandnewCCPbuildingat Vestec. Second part is located at Krc campus. The animalfacilityinKrccontains3separatedbuildingswhichoffertousemice breeding and production barrier, mice experiment andinfectiousBSL2miceexperimentandquarantine.AllKrcsiteshavethesinglecorridordesignandareequippedwithbothIVC’sandopencages,steamsterilisatorsandH2O2decontaminationschambers.Theconstructionandmaintenanceproblemsofthisbuildingsmakeusveryproudofthefactthatallbarrierfacilitiesat Krc campus aremore than 8 years specific pathogen freeaccordingtoFELASArecommendations.

The design of animal facility at Vestec focuses on protectionof animal health due separation of clean and dirty corridorsinsideandoutsidethebarriersandduesandwichstructureoftechnicalandanimalfloors.Thisbrand-newbuildingwithtwounderground and two upper levels houses animals primarilyon both 2nd underground and 2nd upper level and contains 5individual, fully separated breeding and experimental barrierareas,whichcanbe linkedtogether ifnecessary.Eachbarrierincludesmoderndevices likebigvolumesteamsterilizersandH2O2 chambers to sterilize all kinds ofmaterials, air andwetpersonal sluices and pass through boxes, modern and eco-friendlyHVAC technology. All of this important devices helpstokeep the“clean”sideof thebarrier specificpathogen freeaccording to FELASA recommendations. The animals areprotected again at the cage levelwith Individually ventilatedcagesandwithstate-of-the-artDigitalventilatedcages(Fig.1).Digitalventilatedcagessignificantlyincreasetheanimalwelfarelevelandanimalfacilityefficiency,buttheyarealsousefulforresearch as they increase the detectionof strangebehaviour(sick orwounded animals), reduce animal stress and providecontinuousinformationaboutanimalactivity.

Jan HonetschlägerAnimal Facility Module

Animal Facility Module: Taking care of Mouse models

In the Spotlight

Figure1.DigitalVentilatedCageRackfromTecniplast


All technical equipment including HVAC, centralized washingarea, water plant including waste disposal and beddingdispensingvacuumsystemtoincreasethein-housebiosecurityare located in two levels in the middle of the building.This building structure give us the possibility to do mostmaintenance services including hepafilter exchange, controlof air flow and decontamination of animal holding roomsanytimewithoutenteringthebarriers.Moreovershortdistancebetweenthetechnologyandanimalholdingroomdecreasetherisk of contamination of hepafiltered air during its transportto barriers. The quarantine is technologically and personallyfullyseparatedfromotherbarriersand isequippedwithowncage washer, steam and H2O2 decontamination. The flow ofIVC’s andpotentially contaminatedmaterial is restrictedonlywithinthequarantinearea.Exitforpersonnel ispossibleonlythroughwetshowerwithclothesexchange,formaterialsafterdecontamination only. High standard of both Krc and Vesteccampus,responsiblestuff,specificprocedureandtechnologyofpackagingandtransportinganimals,allowustomoveanimalsfromonecampustoanotherandbetweenthebarrierswithoutalteringtheirhealthstatus.

Asmentionedabove,thebuildingandequipmentitselfdoesn’tbringthequalityofanimalfacilityservice.That’swhywefocusoneducationofourpersonneltoo.Werunaninternalcontinuingeducationprogrammeforallfacilityemployees,wheretheycanlearnandimprovetheirskillsandshareexperiencewithothercolleagues(Fig.2).Ascooperationwithprivatecompanieswedo organize seminars for broad spectrum of attendees, givelectures at universities and give practical training to selectedstudents of local high-schools.We do believe that with highlevel motivated and attracted personnel all tasks, SOP’s andunexpected situations will be solved easily and with bestpossiblequality.

AllbuildingsofCCPanimalfacilitywithtotalcapacitymorethan65000 animals are together creating comprehensive solutionformice and rats breeding and experiment.Great advantageofdifferentbuilding locationandtheirdividing into individualsections with high responsibility of our stuff give us theflexibilityforemergencysolutionsandabilitytokeepallanimalshealth protected. Conception, philosophy and animal facilitymanagement is in accordance with highest world standardsfor laboratory animals and is leading to get internationalrespected AAALAC accreditation (Association for assessmentandaccreditationoflaboratoryanimalcare).

In the Spotlight

Figure 3. Laminar Flow technology changing station fromTecniplast.

Figure2.TrainingsessionforAFM’sanimalcaretakers.


Short history of PWD/Ph and PWK/Ph mouse inbred strainsProf. Jiri Forejt

Whatisspecialaboutthesemice?

ThePWD/PhandPWK/Ph(abbreviatedPWDandPWK)strainshavebeenthefirstinbredstrainsderivedfromwildmiceoftheMus musculus musculussubspecies.Mostofthe470laboratoryinbredstrains1originatedfromothermousesubspecies.

Is it important to know what subspecies an inbred strainoriginatedfrom?

Fora geneticist, the intersubspecific variation in thegenomicsequence is an important source of phenotypic variations.The frequencyof single-nucleotide variations, SNVs, betweenmousesubspeciesisanorderofmagnitudehigherthanbetweenindividuals within the same subspecies or, for comparison,betweenhumanpopulations.

Howwerethesemousestrainsprepared?

Westartedin1972withmorethan15pairsofmaleandfemalewildmicefromvariouslocalitiesnearPrague.Toachieve>98.7%homozygosity of the genome, it was necessary to proceedthrough21generationsofbrother x sister crosses. It tookussevenyearstocompletethebreedingplan,buttheyieldwasonlythreestrains,twoofwhich,PWD/PhandPWK/Ph,arestillbreedingandpotentiallyimmortal.

WhatdoesPWD/Phstandfor?

PWDstandsfor`PragueWild-derived,seriesD.Phusedtobean indicator of any mouse strain maintained for at least 20generationsinPrague.

Are these Prague Wild-derived mouse strains used outsidePrague?

Ofcourse,theyareusedinmanylaboratoriesaroundtheworld.WemaintainthemattheInstituteofMolecularGeneticsASCR,butwealsodepositedtheminthelargestreferencerepositoryof the laboratory mice, The Jackson Laboratory (Bar Harbor,MaineUSA).Wedonotpossessdataonthedistributionofourmice fromthe JacksonLab,butour laboratory shipped thesemiceorconsomicstrainsderivedfromthemtomanyacademicinstitutionsintheUSA,GreatBritain,France,GermanyorChina.We also know that these mice or their descendants live inlaboratoriesinIsraelandinAustralia.

Whichofyourdiscoveriesarerelatedtothesemousestrains:

Several achievements of my laboratory in the last 30 yearswouldnotbepossiblewithoutthesemice.Someofthemarelistedbelow:

- Genetic analysis of genomic imprinting of Insulin-like Growth Factor 2 Receptor (Igf2r)

The Thp deletion acts as dominant embryonic lethal whentransmitted from the female but not male parent. Denise

Barlow and coworkers identified the Igf2r gene as the firstmouse imprinted gene and showed that it is deleted in Thp mice2.We found thatembryoswithmaternalThpwere viableif themaleparentwasPWDorPWK. Because Igf2rwas stilltranscriptionally silenced in these embryos, we predicted(wrongly)thatagenecloselylinkedbutdifferentfromIgf2r isresponsibleformaternalThpviability3.

- Mouse consomic strains: exploiting the genetic divergence between Mus m. musculus and Mus m. domesticus subspecies

To exploit the genomic divergence between PWD andclassical laboratory strains for dissection of quantitative,polygenicphenotypeswepreparedapanelof27chromosomesubstitution(consomic)strains,whereindividualchromosomesof the C57BL/6J (M. m. domesticus) strain were substitutedby homologous chromosomes, or their parts, from PWD(M. m. musculus)mice4. These strains continue to serve as auniversaltoolinthestudiesofgeneticcontrolofawidevarietyof phenotypes, including behaviour, bone shape evolution,variations in insulin secretion, virus resistance, and manyothers5. The PWD strain and PWD-derived consomic strainshave been instrumental in our studies of genetic control ofhybridmalesterilityandmeioticrecombinationrate(seemorebelow).

- Participation in the Collaborative Cross Project

PWKwasoneoftheeightstrains,outofover400extantmousestrains,selectedbytheInternationalComplexTraitConsortiumto build a series or recombinant inbred strains poised to‘revolutionize our understanding of system-wide geneticinteractionsenablinganewdisciplinecalled“SystemsGenetics”’6.Originally,thegoalwastoconstruct1000CCstrainstoachieveasingle-generesolutionofgeneticmapping.However,duetoan unexpectedly high incidence of epistatic incompatibilitiesresultinginembryoniclethalityorinfertility,only75completedstrainsareavailableatpresent,andthegoalwasreducedto300strains(http://compgen.unc.edu/wp/?page_id=99).

- Identification of Prdm9, the first hybrid sterility gene in vertebrates

Thebeginningofthisprojectpredatedandinitiatedconstructionof thePWKandPWD inbred strains.Originally,weusedwildmalemicefromdifferentlocalitiesinCzechoslovakiaandfromEurope in crosses with C57BL/10SnPh or C3H/Di laboratoryfemales.Differentwildmalesproduced sterileor fertilemaleprogeny (females were always fertile) with C57BL/10SnPh,butonly fertileprogenywithC3H/Di.Thedifferent fertilityofC57BL/10SnPhandC3H/Dihybridswasgeneticallymappedtoa single genetic locus,Hybrid sterility 1 (Hst1), at the linkagegroup IX (Chromosome17)7.High-resolutiongeneticmappingandbeginningofphysicalmappingofHst1hadtowaitfor20yearsbeforethemolecularcloningtechnologybecameavailableforthemouse.Then,usingpositionalcloningandoverlapping

In the Spotlight


In the Spotlight

BAC clones from the C3Hmouse genome as transgenes, weachieved fertility rescue of sterile hybrids and identified theHst1 locus with the PR domain-containing 9 (Prdm9) gene8.Theearlystagesofgeneticstudiesofhybridsterilityhavebeenreviewed9-11. One year after the discovery of Prdm9 as thehybridsterilitygene,threepapersreportedtheroleofPrdm9 inmeioticrecombinationbydeterminingthegenomiclocationof recombination hotspots12-14. This was the first indicationthathybridsterilitycouldbe linkedtomeioticrecombination.Recombinationintheformofcrossoversandgeneconversions(noncrossovers)betweenhomologouschromosomesincreasesallelic combinations in a population, enables pairing andsynapsis of homologs at the firstmeiotic prophase and theirpropersegregationintogametes.ThemajorfailureofmeiosisIn(PWDxB6)sterilehybridsconsistsinincompletemeioticpairingandsynapsisofhomologscomingfromdifferentsubspeciesandassociated failureofproper transcriptional inactivationof sexchromosomes. Next, the secondmajor hybrid sterility factor,Hstx2wasmappedon theX chromosome15-17.Wespeculatedthattheprimarycauseofmeioticarrestinhybridscouldbetheincreasedgenomicdivergencebetweenbothsubspecies,whichcould hamper recognition of homologous chromosomes andresult in consequent failure of their synapsis16. SimonMyers’groupadoptedourmodelandanalyseddistributionofmeioticrecombinationhotspots in(PWDxB6)malesusingchromatinimmunoprecipitation of DNA sequences associated with theDMC1single-strandrecombinationprotein.Theyfoundstrong

asymmetryofDNAdouble-strandbreakhotspots.ThePRDM9bindingsitesofPWDoriginwerepreferentiallyfoundonC57BL/6homologsandviceversa.Theauthorslinkedthisasymmetrytothefailureofhomologstopairproperly18.Inaseparatestudy,Camerini-Otero’s group compared series of different hybridsincluding our (PWDxC57BL/6)F1 sterile males and concludedthatthemeioticarrestandhybridsterilityisassociatedwiththeoccurrenceofanomalouspromoter-associatedDMC1hotspotstypicalofPRDM9nullmales19.Thus,furtherstudiesareneededtoclarifythemolecularmechanismofPrdm9actioninhomologchromosomepairingandhybridsterility.

- Genetic control of genome-wide recombination rate

To further elucidate the relationship between hybrid sterilityandgeneticrecombinationweanalysedtheeffectofindividualPWDchromosomesongenome-wide recombination rate.Weused the C57BL/6-Chr #PWD panel of 27 consomic strains tocomparethefrequencyofcrossovers,monitoredasMLH1focionsynaptonemalcomplexesofpachytenespermatocytesandoocytes, and compared them to parental PWD and C57BL/6controls. Surprisingly, Prdm9 itself had no effect on thecrossover frequency,but thestrongest locusaffectingmeioticrecombinationratewasidenticalwiththesecondmajorhybridsterility locus, Hstx2. Thus both, Prdm9 and Hstx2 hybridsterility genesareassociated, atdifferent levels,withgeneticrecombination20.

Full Name Abbreviated name Available from Comment 1

IMG AS CR JAX*

C57BL/6J-Chr1PWD/Ph/ForeJ B6.PWD-Chr1 X X

C57BL/6J-Chr2PWD/Ph/ForeJ B6.PWD-Chr2 X XC57BL/6J-Chr3PWD/Ph/ForeJ B6.PWD-Chr3 X XC57BL/6J-Chr4PWD/Ph/ForeJ B6.PWD-Chr4 X ?C57BL/6J-Chr5PWD/Ph/ForeJ B6.PWD-Chr5 X XC57BL/6J-Chr6PWD/Ph/ForeJ B6.PWD-Chr6 X XC57BL/6J-Chr7PWD/Ph/ForeJ B6.PWD-Chr7 X XC57BL/6J-Chr8PWD/Ph/ForeJ B6.PWD-Chr8 No XC57BL/6J-Chr9PWD/Ph/ForeJ B6.PWD-Chr9 X XC57BL/6J-Chr10.1PWD/Ph/ForeJ B6.PWD-Chr10.1 No XC57BL/6J-Chr10.2PWD/Ph/ForeJ B6.PWD-Chr10.2 X XC57BL/6J-Chr10.3PWD/Ph/ForeJ B6.PWD-Chr10.3 X XC57BL/6J-Chr11.1PWD/Ph/ForeJ B6.PWD-Chr11.1 X X PWDsequencecentromere-79.6MbC57BL/6J-Chr11.2PWD/Ph/ForeJ B6.PWD-Chr11.2 X X PWDsequence44.0-96.6MbC57BL/6J-Chr1013PWD/Ph/ForeJ B6.PWD-Chr11.3 X X PWDsequence79.8-telomereC57BL/6J-Chr12PWD/Ph/ForeJ B6.PWD-Chr12 X XC57BL/6J-Chr13PWD/Ph/ForeJ B6.PWD-Chr13 X XC57BL/6J-Chr14PWD/Ph/ForeJ B6.PWD-Chr14 X XC57BL/6J-Chr15PWD/Ph/ForeJ B6.PWD-Chr15 X XC57BL/6J-Chr16PWD/Ph/ForeJ B6.PWD-Chr16 X XC57BL/6J-Chr17PWD/Ph/ForeJ B6.PWD-Chr17 X X

Table1.Completelistofconsomicstrainsandtheiravailability.

* The photo of Professor Forejt was taken by Martin Kovář.


- What are the ongoing projects involving PWD and PWK mice?

Several joint projects, all based on informal collaborations,areinprogress.WiththelaboratoryofLindaOdenthal-Hesse,MaxPlanckInstitute,Ploen,Germany,andwithJaroslavPialek,Institute of Vertebrate Biology, Studenec, we identify newPrdm9allelesinwildmicefromdifferentpopulationsaroundtheworldandchecktheireffectonthefertilityofintersubspecifichybrids.WeparticipateintheprojectofJosephNadeau,PacificNorthwestResearchInstituteSeattle,USA,aimedtodeterminethe mouse genes controlling the composition of microbiota,using our C57BL/6-Chr#PWD consomic mice. Finally, wecollaboratewiththebiotechcompanyGenentech,amemberoftheRochegroup,SanFrancisco,USA.

- Did you expect such a wide use of your strains 40 years ago, when you started their preparation?

Do you know how we began 40 years ago? We were notpermitted(forgoodreasons)tobringwildmicetotheroomsoftheanimalfacility,soweoccupiedadefunctlavatoryinthebasementofthebuilding,installedtwostandsformousecages,andstartedbreeding.Iwastoldthatweweredoingafutilejobbecause wildmice cannot breed in captivity. Indeed, at thattime,noonecould image thatonedaydescendantsof thesemicewouldliveinmanylaboratoriesofthefivecontinents.

In the Spotlight

References1. BeckJ,LloydS,HafezparastM,Lennon-PierceM,EppigJ,etal.(2000)Genealogiesofmouseinbredstrains.NatGenet24:23-25.2. BarlowD,StogerR,HerrmannB,SaitoK,SchweiferN(1991)Themouseinsulin-likegrowthfactortype-2receptorisimprintedandcloselylinkedto

theTmelocus.Nature349:84-87.3. ForejtJ,GregorovaS(1992)Geneticanalysisofgenomicimprinting:anImprintor-1genecontrolsinactivationofthepaternalcopyofthemouseTme

locus.Cell70:443-450.4. GregorovaS,DivinaP,StorchovaR,TrachtulecZ,FotopulosovaV,etal.(2008)Mouseconsomicstrains:exploitinggeneticdivergencebetweenMusm.

musculusandMusm.domesticussubspecies.GenomeRes18:509-515.5. NadeauJH,ForejtJ,TakadaT,ShiroishiT(2012)Chromosomesubstitutionstrains:genediscovery,functionalanalysis,andsystemsstudies.Mamm

Genome23:693-705.6. ChurchillGA,AireyDC,AllayeeH,AngelJM,AttieAD,etal.(2004)TheCollaborativeCross,acommunityresourceforthegeneticanalysisofcomplex

traits.NatGenet36:1133-1137.7. ForejtJ,IvanyiP(1974)Geneticstudiesonmalesterilityofhybridsbetweenlaboratoryandwildmice(MusmusculusL.).GenetRes24:189-206.8. MiholaO,TrachtulecZ,VlcekC,SchimentiJC,ForejtJ(2009)AmousespeciationgeneencodesameiotichistoneH3methyltransferase.Science323:

373-375.

Full Name Abbreviated name Available from Comment 1

IMG AS CR JAX*

C57BL/6J-Chr18PWD/Ph/ForeJ B6.PWD-Chr18 X XC57BL/6J-Chr19PWD/Ph/ForeJ B6.PWD-Chr19 X XC57BL/6J-Chr19.REK5PWD/Ph/ForeJ B6.PWD-Chr19.REK5 X No PWDsequence50.1Mb-telomereC57BL/6J-Chr19.REK8PWD/Ph/ForeJ B6.PWD-Chr19.REK8 X No PWDsequence32,93Mb-telomereC57BL/6J-ChrX.1PWD/Ph/ForeJ B6.PWD-ChrX.1 X X PWDsequencecentromere-64.9MbC57BL/6J-ChrX.1sPWD/Ph/ForeJ B6.PWD-ChrX.1s X No PWDsequencecentromere-69.6MbC57BL/6J-ChrX.2PWD/Ph/ForeJ B6.PWD-ChrX.2 X X PWDsequence38.5-129.4MbC57BL/6J-ChrX.3PWD/Ph/ForeJ B6.PWD-ChrX.3 X X PWDsequence98.25Mb-telomereC57BL/6J-ChrYPWD/Ph/ForeJ B6.PWD-ChrY X XC57BL/6J-mtPWD/Ph/ForeJ B6.PWD-mit X X

Figure1.A)PWD/Ph(agouti)mouseandB)C57BL/6Jmouse(black)


9. ForejtJ(1996)Hybridsterilityinthemouse.TrendsGenet12:412-417.10. ForejtJ(2013)Hybridsterility,Mouse.In:BrennerS,editor.EncyclopediaofGenetics.NewYork:AcademicPress.11. ForejtJ,PialekJ,TrachtulecZ(2012)Hybridmalesterilitygenesinthemousesubspecificcrosses.In:MacholanM,BairdSJE,MuclingerP,PialekJ,

editors.EvolutionoftheHouseMouse.Cambridge:CambridgeUniversityPress.12. BaudatF,BuardJ,GreyC,Fledel-AlonA,OberC,etal.(2010)PRDM9isamajordeterminantofmeioticrecombinationhotspotsinhumansandmice.

Science327:836-840.13. MyersS,BowdenR,TumianA,BontropRE,FreemanC,etal.(2010)DriveagainsthotspotmotifsinprimatesimplicatesthePRDM9geneinmeiotic

recombination.Science327:876-879.14. ParvanovED,PetkovPM,PaigenK(2010)Prdm9controlsactivationofmammalianrecombinationhotspots.Science327:835.15. BhattacharyyaT,GregorovaS,MiholaO,AngerM,SebestovaJ,etal.(2013)Mechanisticbasisofinfertilityofmouseintersubspecifichybrids.ProcNatl

AcadSciUSA110:E468-477.16. BhattacharyyaT,ReifovaR,GregorovaS,SimecekP,GergelitsV,etal.(2014)Xchromosomecontrolofmeioticchromosomesynapsisinmouseinter-

subspecifichybrids.PLoSGenet10:e1004088.17. Dzur-GejdosovaM,SimecekP,GregorovaS,BhattacharyyaT,ForejtJ(2012)Dissectingthegeneticarchitectureoff(1)hybridsterilityinhousemice.

Evolution66:3321-3335.18. DaviesB,HattonE,AltemoseN,HussinJG,PrattoF,etal.(2016)Re-engineeringthezincfingersofPRDM9reverseshybridsterilityinmice.Nature

530:171-+.19. SmagulovaF,BrickK,PuYM,Camerini-OteroRD,PetukhovaGV(2016)Theevolutionaryturnoverofrecombinationhotspotscontributestospeciation

inmice(vol30,pg266,2016).Genes&Development30:871-871.20. BalcovaM,FaltusovaB,GergelitsV,BhattacharyyaT,MiholaO,etal.(2016)HybridSterilityLocusonChromosomeXControlsMeioticRecombination

RateinMouse.PlosGenetics12.doi:ARTNe100590610.1371.

In the Spotlight

CzechCentreforPhenogenomicswouldliketocongratulate

Professor Jiři Forejt

whowasrecentlyawarded

The National Prize of the Czech Government “Česká hlava” (Czech Brains)

forhislifelongresearchinthefieldofmousegenetics

* official image from Česká Hlava awards


Careers

CCP comprises a young, multidisciplinary and international team. We believe in the personal and professional development of our staff and seek, where possible, to facilitate the attendance of relevant conferences and courses. We offer a competitive salary and various working contracts. For a full list of our available positions please visit our website www.phenogenomics.cz.

Allpositionsareavailableimmediatelyforaninitialfixed-termcontract(1year),withpossiblelonger-termextensionupondemonstratedproficiency.Formoreinformationortoapplyforanyofthesepositions,contactMrLiborDanek([email protected]).AllapplicationsshouldbemadeinEnglish,includealetterofinterestandastructuredCV.

Junior Research Position:Phenotyping Pipeline (Vision)The current open position is for vision screen & research unitfocusedonthestandardizedphenotypingofgeneratedtransgeniclineswithaimtorevealandannotateunknownfunctionsofgenesfromsystematicproductionofKOlineswithinInternationalMousePhenotyping Consortium. The workflow of the unit consists ofstandardized measurement focused on eye morphology andfunction. The junior researcher will also have the opportunityto carry out his/her own research project on eye physiology,morphology, or development areas. The attendance of relevantscientificmeetingsandconferencesisalsoencouraged.

The idealcandidate isa recentPhDgraduate in therelevantfieldwithstrongplanning,organizationalandproblemsolvingskillsandwithstructuredapproachtodatacollectionandanalysis.

Research position (postdoc) in ubiquitin ligasesApostdoctoralresearchscientistpositionisavailableimmediatelyintheLaboratoryofTransgenicModelsofDiseasesattheInstituteofMolecular Genetics of the ASCR, v.v.i. located in BIOCEV (CCPbuilding)inVestecnearPrague,CzechRepublic.

Weare looking forahighlymotivated individualwithastrongbackgroundandskillsetinbiochemistryandmolecularbiology.Youshould have a Ph.D. in biology or biochemistry and have a goodproficiencyinEnglish.Experienceinproteinbiochemistryisaplus.Yourworkwill focus onwet bench projects including in vitro, invivo,andmousemodelswiththegoalofelucidatingthemolecularmechanismsofubiquitinationmediatedbyRING-typeE3ubiquitinligases.

Laboratory Technicians:Phenotyping ScreensThe Czech Centre for Phenogenomics is seeking talented andmotivatedtechnicalassistantstoworkaspartofourphenotypingmoduleforvariousscreens(vision,hearing,behavioretc)tobecomepartofaninternationalteamdedicatedtothehigheststandardsofpre-clinicalandbasicbiologicalresearch.

YoushouldpossesspreferablyatleastBScinbiologicalormedicalsciences (or related field) and have a good command of English(spokenandwritten).Becausetheworkincludesdailyinteractionswith laboratory animals previous rodent handling experience orvalidcertificationforanimalhandlingwouldbeadvantageousbutisnotessential.Youshouldbeskilful,wellorganizedandcapableofworkingaspartofateam.

Successful candidates will work primarily in one unit, however,therewillbeopportunitiesforcrosstrainingwithotherunitswithinthePhenotypingModule.

Pathologist (Rodent Pathology)Toadvanceandfurtherimproveservicesofourhistopathologylabwe are seeking experienced pathologist whowill be responsiblefor analyses and descriptions of mouse and rat tissue samples,especially:

• to provide expertise in the pathology of genetically-engineeredmouse(GEM)andratmodels

• to provide full pathology analysis including completegross- and histopathological evaluation suppliedwith image-based report, digital images, andrecommendations

• toperformphenotypeinvestigationandcharacterizationtogether with histology-lab managing scientist; thisincludes necropsy, macroimaging, tissue samplecollection, supervision of histological processing,histopathologicalevaluation,digitalphotomicrographs,andconsultations.

• tofollowandimplementGLPrulesandmanageworkoflabtechnicians

• to drive his/her own research projects and activelyparticipatewithintheotherprojectsoftheCentre.

Successful applicant should have DVM or MD (or equivalentadvanced degree in relevant field) and relevant research and/or hands-on experience. Capability to work in English speakingenvironment isamust,previousexperiencewithSOPs forGLP isanasset.

To view all available positions, visit our website www.phenogenomics.cz


PaulaJPdeVreeetal,Targetedsequencingbyproximityligationforcomprehensivevariantdetectionandlocalhaplotyping.NatureBiotechnology,32,1019–1025(2014)

KnutJEgelieetal,TheemergingpatentlandscapeofCRISPR-Casgeneeditingtechnology.NatBiotechnol.34(10):1025-1031(2016)

HikabeO,etal,Reconstitutioninvitrooftheentirecycleofthemousefemalegermline.Nature.539(7628)299-303(2016)[Epubaheadofprint]

Wilhelmetal.FOXO1couplesmetabolicactivityandgrowthstateinthevascularendothelium.Nature529,216-220(2016).

Upcoming Events

2nd Programmable Nucleases (CRISPR Cas9) Transgenesis Course

3rd-7thApril2017|Vestec,CzechRepublic

www.phenogenomics.cz

CITIM 2017 – CANCER IMMUNOTHERAPY & IMMUNOMONITORING International Congress in Prague

24.4.2017-27.4.2017|

http://www.canceritim.org/index.html

Metabolic disorders and liver cancer

23–26April2017|ES–PalmadeMallorca

EMBOWorkshop

http://meetings.embo.org/events/17-brown-fat

Advances in transgenic animal models and techniques

11th-12thMay2017|Nantes,France

http://www.trm.univ-nantes.fr/

9TH Workshop on Innovative Mouse Models

June 15 - 16, 2017| Leiden, The Netherlands

http://research.nki.nl/immworkshop/

Mouse genome engineering

10th–20thJuly2017|Dresden,Germany

http://meetings.embo.org/event/17-mouse-genome

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