Neurons

TobaccoplantwithGFP

TRANSGENICS

GloFish

Overview

Historicbackground Conceptstoremember Transgenictechnologyandtransgenicanimals Applica>onsoftransgenicanimals Construc>onoftransgenicanimals Ethicalconcerns

Historicalbackground
Observa>onofinheritedcharacteris>csorspontaneousmuta>ons. Selec3vebreedingwasacommonprac>ceamongfarmersfortheenhancementofchosen traits,e.g.,increasedmilkproduc>on. 1970s:Firstchimericmicewereproduced(Brinster,1974).Thecellsoftwodierent embryosofdierentstrainswerecombinedtogetheratanearlystageofdevelopment (eightcells)toformasingleembryothatsubsequentlydevelopedintoachimericadult, exhibi>ngcharacteris>csofeachstrain. 1981:DNAmicroinjec3on,thersttechniquetoprovesuccessfulinmammals,wasrst appliedtomice(GordonandRuddle)andthentorats,rabbits,sheep,pigs,birds,andsh. ThetermtransgenicwasrstusedbyJ.W.GordonandF.H.Ruddle(hadrapid developmentintheuseofgene>callyengineeredanimalswithanincreasingnumberof applica>onsforthetechnology). 1986:Retrovirusmediatedtransgenesis(Jaenisch,1986) Embryonicstem(ES)cellmediatedgenetransfer(Gossleretal.,1986) Fastdevelopmentandrou3nelyusedlabtechniqueinresearch,nowadays.

Conceptstoremember

PlantoranimalDNA

RecombinantDNA technology

Specic restric>on enzyme DonorDNA Engineeredplasmid incorporatesintobacteria whichreproduceand clonethegenefrom donorcellthatwasspliced intoplasmid

Bacterialplasmid
Eachplasmid contains adierent donorDNA fragment

Specic restric>on Enzyme (same) PlasmidDNA (vector)

S>ckyends combine (complementary)

Conceptstoremember
DNA break

HomologousRecombina3on

Duringmeiosisandmitosiswhen Protein Resection 5 ends are cut away homologouschromosomesalignalong complex Rad52 themetaphaseplane,recombina>on takesplacewithinthehomologous sequences.Recombina>onmaytake placeanywherewithintheankingDNA Strand invasion at homologous sites sequencesandtheexactloca>onis determinedbythecells.Theendresult isanewpieceofDNAinsertedintothe chromosome.Therestofthegenomeis DNA synthesis unaltered.

Applica3on:Toreplaceoneallelewithan engineeredconstructbutnotaectany otherlocusinthegenome WemustknowtheDNAsequenceof thegenewewanttoreplace

Transgenictechnology Whataretransgenicanimals?
Gene>cmanipula>onofanorganism(animalorplant)whichpermitsstable integra3onandexpressionofexogenousDNAfragmentsintothegenomeof theorganism. Inthistalk,thetermtransgenicanimalincludes: animalsthatcarryforeignDNArandomlyintegratedintotheir genomes animalsgeneratedbyhomologousrecombina>on,allowing theresearchertocontroltheloca3onoftheinsertedDNA (includeKOsendogenousgenehasbeenspecically inac>vatedKisgeneofinteresthasbeenaddedtothe genomeorana>vegenehasbeenenhanced)

Applica3onsoftransgenicanimals
Medicalresearch:iden>fythefunc>onsofspecicfactorsincomplexhomeosta>csystems throughoverorunderexpressionofamodiedgene(theinsertedtransgene);modelsof humandiseaseprocesses Toxicology:responsivetestanimals(detec>onoftoxicants); Developmentalgene3cs; MolecularBiology:theanalysisoftheregula>onofgeneexpressionmakesuseofthe evalua>onofaspecicgene>cchangeatthelevelofthewholeanimal; Pharmaceu3calIndustry:targetedproduc>onofpharmaceu>calproteins,drugproduc>on andproductecacytes>ng; Biotechnology:producersofspecicproteins;gene>callyengineeredhormonestoincrease milkyield,meatproduc>on;gene>cengineeringoflivestockandinaquacultureaec>ng modica>onofanimalphysiologyand/oranatomy;cloningprocedurestoreproduce specicbloodlines; Developinganimalsspeciallycreatedforuseinxenogra[ing.

Construc3onoftransgenicorganisms
1)DeliveryofDNA
Transfec3on(chemical,liposome mediated,electropora>on) Transduc3on(highleveltransient expression,longtermstable expression,stabletransforma>on) Directtransfer(microinjec>on,par>cle bombardment) Nucleartransfer/Stemcelltransfer Otherexamples: Pelementtransforma3on; RecombinantTiplasmid

2)Designtheconstructaccordingtothestrategy
Transgenesis: Aimofgenetransferstudiesinuences designofconstruct Transgeneexpression Transgenepromoter:inducibleexpression Genetarge3ng: Condi3onalgenemodica3ons:CreloxP SinglegeneknockoutsandKnockins Geneknockdown(RNAi) Reportergenes(GFAPtagging)

Construc3onoftransgenics:deliveringDNAintocells
TRANSFECTION Chemicaltransfec3on:calciumphosphateor dextransulfateopenstransientholesinacells membrane,admibngreplacementDNA Liposometransfer:liposomecarriesageneintoa soma>ccellwherethedeliveredgenemay replaceanormalone Electropora3on:electricalcurrentopenstransient holesinacellsmembrane,admibngreplacement DNA DIRECTPHYSICALTRANSFER Microinjec3on:>nyneedleinjectsDNAintoa celllackingthatDNAsequence Par3clebombardment:metalpellets(goldor tungsten)coatedwithDNAareshotwith explosiveforceorairpressureintorecipient cells TRANSDUCTION Virus:humangeneinsertedintoaherpesvirus whichinfectsahumancell,whereitisexpressed Retrovirus:RNAviruscarryingRNAversionof humangeneinfectsasoma>ccell.Thegeneis reversedtranscribedtoDNAandinsertsintoa humanchromosome.Hereitmayproducea missingorabnormalprotein NUCLEARTRANSFER Transferofasoma3ccellnucleusintoan enucleatedegg.Theeggiss>mulatedwithashock andacermanymito>cdivisions,thissinglecell formsablastocystwithalmostiden>calDNAto theoriginalorganism. STEMCELLTRANSFER Transfer of embryonic stem cells into a developing embryo by microinjec>on into blastocysts. The resultanttransgenicanimalpossessesapropor>onof cellsdescendedfromtheEScelllinage.

Construc3onoftransgenics:deliveringDNAintocells
TRANSFECTION (1)Chemical

Mammaliancellsinculture

Lowcost Cellswithinserted DNAcanbeselected byampicillin

Construc3onoftransgenics:deliveringDNAintocells
TRANSFECTION (2)Liposomemediated

Highlyecient Liposomehasamembrane similartothecellallowingittofuse withtherecipientcellmembrane, releasingtheDN.A

Construc3onoftransgenics:deliveringDNAintocells
TRANSFECTION (3)Electropora3on

Controlledmilisecondelectricalpulsesareappliedtotheneedleelectrode,whichform anelectriceld,openingholesinthecellmembrane,allowingDNAtoenterthecell

Construc3onoftransgenics: deliveringDNAintocells
TRANSDUCTION
ProcesswherebyforeignDNAisstably introducedintoanothercellviaaviral vector.

Construc3onoftransgenics:deliveringDNAintocells
Retrovirusmediatedtransfer

Construc3onoftransgenics:deliveringDNAintocells
DIRECTPHYSICALTRANSFER Microinjec3on Par3clebombardment

Construc3onoftransgenics:deliveringDNAintocells
SOMATICCELLTRANSFER/Pronucleartransfer

The transgene can integrate immediately (mouse is transgenic) but more common the DNA to integrate acer one or two cell divisions,inwhichcasetheresul>ngmouseis a mosaic containing both transformed and nontransformedcells. Newbornmiceresul>ngfromdevelopment oftheimplantedembryosarecheckedby PCRorSouthernblo>ngoratestfor tansgeneexpressionforthepresenceofthe desiredDNAsequence. Theywillbeheterozygousforthedesiredgene(transgenicfounder).

Eciencyofmicroinjec3on

Nomorethan10%ofmiceprogenywillbetransgenic.Whilethetechniquecanbeapplied toothermammals,thetransgenicprogenyismuchlower(<1%).Thisispartlyduetothe dicultyinhandlingeggs,andpartlyduetothelowersurvivalrates.

Construc3onoftransgenics:deliveringDNAintocells
ESTRANSFER

TheCellChapter3

Construc3onoftransgenics:deliveringDNAintocells
ESTRANSFER

Ecienttransfec>on Posi>venega>veselec>on Genetarge>ngorinser>onofthegene Characteriza>onbeforeanimalgenera>on Timeandcostintensive

Construc3onoftransgenics:deliveringDNAintocells
RecombinantTiplasmidintegra3oninPlants

protoplast

Construc3onoftransgenics:deliveringDNAintocells
Pelementtransforma3onDrosophilamelanogaster(1)
HighlymobileDNAelement,which cantransposefroman extrachromosomalelementintoa chromosome.Generally,this procedureresultsinincorpora>onof asinglecopyofthetransgeneinto theDrosophilagenome.Incontrast, transgenicmicecarrymul>plecopies ofthetransgeneincorporatedinto theirchromosomes.Inboth organisms,chromosomalinser3onis highlyvariable. Individualscarryingthetransgene arerecognizedbyexpressionofa markergene(ex:eyecolor)thatis alsopresentonthedonorDNA.

Construc3onoftransgenics:deliveringDNAintocells
Pelementtransforma3onDrosophilamelanogaster(2)
Fliesthatdevelopfrominjected embryoswillcarrysomegermcells thathaveincorporatedthe transgene:someoftheprogenywill carrythetransgeneinallsoma>c andgermlinecells,givingriseto puretransgeniclines.Individuals carryingthetransgeneare recognizedbyexpressionofa markergene.Althoughthe transgenesinDrosophilaandmice insertinchromosomalsitesdierent fromtheposi>onofthe correspondingendogenousgene, theyusuallyareexpressedinthe right>ssueandattheright>me duringdevelopment.

Construc3onoftransgenicorganisms
1)DeliveryofDNA
Transfec3on(chemical,liposome mediated,electropora>on) Transduc3on(highleveltransient expression,longtermstable expression,stabletransforma>on) Directtransfer(microinjec>on,par>cle bombardment) Nucleartransfer/Stemcelltransfer Otherexamples: Pelementtransforma3on; RecombinantTiplasmid

2)Designtheconstructaccordingtothestrategy
Transgenesis: Aimofgenetransferstudiesinuences designofconstruct Transgeneexpression Transgenepromoter:inducibleexpression Genetarge3ng: Condi3onalgenemodica3ons:CreloxP SinglegeneKnockoutsandKnockins GeneKnockdown(RNAi) Reportergenes(GFAPtagging)

Construct/transgenedesign
Aimofgenetransferstudiesinuencesconstructdesign Gainoffunc3ontransgene Addnewfunc3onstorecipientindividual genomicgene cDNAsequence Lossoffunc3ontransgene Geneknockout(Totalinac>va>on) Genetarge>ng(Par>alinac>va>on/changeoffunc>on) CreloxPsystem(Condi>onalinac>va>on) Productcandisrupt/interferewithhostgeneexpression an>senseRNA dsRNA smallinterferingRNA(siRNA) Reportertransgene Studying promoter ac>vi>es (>ssue/developmental stage/ cell/typespecicexpression)

Transgeneexpression

Transgenepromoter

Importantconsidera3onsinconstruct design/genetransferexperiments: Controloftransgeneexpression Structureofthepromoter Op>miza>onoftransla>onalstartsite Inclusionsuitablepep>detarge>ngsignal

Thetransgenepromoterdenes spa3alandtemporal pahernofexpression

Maximumcontrolovertransgene expressioninbothcelllinesand animalsisprovidedbyinducible promoters(switchedonandoby controllingthesupplyofapar>cular chemicalligand). Bylinkingthetransgenetoa suitablecellorstagespecic promoter,thedesiredexpression pahernmaybeachieved.

Transgeneexpressionisregulatedby sequencespresentintheexpression construc3onandalsobyfactorsintrinsicto thehostgenome.

Intransgenicanimals,itisocendesirabletoexpressthetransgeneinpar>cular>ssuesoratpar>culardevelopmentalstages

Constructdesign:promoters
Inducibleexpression:An>bio>cinducible(TetOnTetO)expressionsystem
Cons>tu>veexpression TetRepressor

Inthisexpressionsystem,induc>onoccursattheleveloftranscrip3on (SLOWresponsetoinduc3on).

Constructdesign:promoters
Inducibleexpression:Hormoneinducibleexpressionsystem

Protein(X)fusedwiththeestrogenreceptor(ER)is generally inac3ve sequestered into a complex withheatshockprotein90(Hsp90).

Induc3on is fast: requires only the dissocia>on of a protein complex and not transcrip>onfollowedbyproteinsynthesis.

Constructdesign:condi3onalgeneinac3va3on
CreloxPsystem(1)
Crerecombinase TypeITopoisomerasefromP1bacteriophage thatcatalyzessitespecicrecombina>onof DNAbetweenloxPsites

loxPrecogni3onsequence

Specic34bpsequencesconsis>ngofan8bp coresequence,whererecombina>ontakes place,andtwoanking13bpinvertedrepeats conferingorienta>on.

Constructdesign:condi3onalgeneinac3va3on
CreloxPsystem(2)

TheoutcomeofaCreloxrecombina3onisdeterminedbytheorienta3onandloca3onofanking loxPsites.(A)IftheloxPsitesareorientedinoppositedirec3ons,Crerecombinasemediatesthe inversionoftheoxedsegment.(B)IftheloxPsitesarelocatedondierentchromosomes(trans arrangement),Crerecombinasemediatesachromosomaltransloca>on.(C)IftheloxPsitesare orientedinthesamedirec3ononachromosomesegment(cisarrangement),Crerecombinase mediatesadele>onoftheoxedsegment

Constructdesign:condi3onalgeneinac3va3on
CreloxPsystem(3)
Typically,CreandloxPstrainsaredevelopedseparatelyandcrossedtoproduceaCre loxstrain(Nagy2000).ThemajorityofCreandloxPstrainsbeingdevelopedfallintoone ofthefollowingcategories: *Creexpressingstrains:containatransgenethatexpressescreunderthecontrolofa widespread(general)or>ssuespecic(condi>onal)promoter.Theyareusedtoproduce generalorcondi>onalknockoutsrespec>vely. *InducibleCrestrains:containatransgenethatexpressesamodiedformofCre recombinasethatisnonfunc>onalun>laninducingagent(suchasdoxycycline, tetracycline,RU486,ortamoxifen)isadministeredatadesired>mepointinembryonic developmentoradultlife *LoxPanked(oxed)strains:containloxPsitesanking(oneachsideof)acri>cal por>onofatargetgeneorgenomicregionofinterest *Crereporterstrains:containloxPsitesincombina>onwithvisible(uorescentor lacZ)markerproteinsusedtotraceCrerecombina>onsuccessand/oraltera>onsingene expression.

Constructdesign:condi3onalgeneinac3va3on
CreloxPsystem(4)

MaphiasZepper(Curnen)

Constructdesign
Genetarge3ng
Processofdisrup>ngormuta>ngaspecicgene>clocusinembryonicstem(ES)cells,usuallywith theinten>onofmakingknockoutorknockinmicebyinjec>ngthoseEScellsintoblastocysts. Theen>regenetarge>ngprocessconsistsofthefollowingmajorsteps. 1)Linearizeandpurifythetarge>ngconstruct;introduceitintoEScellsbyelectropora>on;grow clonesunderposi>veselec>onbyan>bio>cs;pickseveralhundredresistantclonesinto96well plates;splitclonesintoduplicatesets;freezeonesetandisolateDNAfromtheotherset. 2)GenotypeallclonesbySouthernblotusingaprobespecicforoneendoftheinsertedDNA. 3)Expandclonesthathavethecorrectgenotype(heterozygousforthetargetedallele),freezeback mul>plevialsofeachclone,andprepareabout50microgramsofgenomicDNAfromeachclonefora secondgenotypingassay(SouthernblotorPCR)tocharacterizetheotherendoftheinsertedDNA. Clonesthatareposi>veforbothgenotypingassaysmaythenbeusedforinjec>onintoblastocyststo makechimericmice.

Constructdesign:genetarge3ng
Genetarge3ng:geneknockout

Inser4onvector method

Replacementvector method
tkconferssensi3vitytoganciclovir

Geneknockoutbyhomologousrecombina3oncaninac>vategenes atapredeterminedlocuswithinanintactcell
.

Constructdesign:genetarge3ng
Genetarge3ng:Geneknockinbyintroduc>onofsubtlemuta>ons
Some genes are cri>cal early in development and simple knockout experiments are generallynothelpfulbecausedeathensuesattheearlyembryonicstage(exNMDA)

Inser3onvectors

Replacementvectors

Subtlemuta3onispresentontherst targe3ngconstruct; Intrachromosomalrecombina>onleadstothe elimina>onofthemarkergeneandvector backbone.

Asecondtarge3ngconstructisusedto replacethemuta>onintroducedbytherst. Thesecondconstructincorporatesacounter selectablemarkeroutsidethehomology regiontoavoidrandomintegra>on.

Constructdesign:genetarge3ng
Genetarge3ng:Knockdown RNAinterference
LongdoublestrandedRNAscanbeusedtosilencethe expressionoftargetgenesinavarietyoforganismsandcell types. Uponintroduc>on,thelongdsRNAsenteracellular pathwaythatiscommonlyreferredtoastheRNA interferencepathway.First,thedsRNAsgetprocessedinto 2025nucleo>desmallinterferingRNAsbyanRNaseIIIlike enzymecalledDicer(ini>a>onstep).Then,thesiRNAs assembleintoendoribonucleasecontainingcomplexes knownasRNAinducedsilencingcomplexes(RISCs), unwindingintheprocess. ThesiRNAstrandssubsequentlyguidetheRISCsto complementaryRNAmolecules,wheretheycleaveand destroythecognateRNA(eecterstep).Cleavageof cognateRNAtakesplacenearthemiddleoftheregion boundbythesiRNAstrand.

Hightoxicityoftransfectedcells; Canhaveeectonotherhostproteins

Constructdesignuseofreportergenestostudyexpression
Genetarge3ng:GFPtaggingtostudyproteinlocaliza>on
A)Construc>onofGFPtagged protein B)TransgenicmicewithGFPfusedtoan epithelialprotein

A)TherecombinantgeneencodesafusionproteinthatcontainsGFPatitsCterminus. B) This mouse contains a GFPlabelled transgene expressed throughout the body; the en>re mouse becomes uorescentwhenilluminatedwithUVlightasatthebopom.Thesamemouseisshowninnormallightatthetop.
Gene>cs:FromgenestoGenomes,2/e.(McGrawHillCompanies,2004)

Timelinefordevelopmentofatransgenicmouseline
Classictransgenic
Week1 PrepareDNA construct Week5 Founderpupsborn Week16 F1pupsborn Week23 Colonyexpansion

Week2 Microinjec3on andimplanta3on

Week8 Founderanimals weanedandgenotyped

Week13 Transgenic foundersmated

Week19 F1sgenotyped germlinetransmission

ESderivedtransgenic
Week1 PrepareKO construct Week5 Apply selec3on Week9 Genotypefor Homologous recombina3on Week12 Transferinto Pseudopregnant female Week18 Chimeras Matedfor wildtype Week22 Kosiden3edby Coatcolor

Week4 Transform EScells

Week7 Expand Transformed cells

Week15 Chimeric Animalsborn

Week21 F1genera3on born

Week27 Beginplanned breeding

Backcrosstofounderfor10genera>onstocleanbackground

Whythemouse?

Whythemouse?

Ofthemodelorganismswhichmaybegene3callymodied,themouseis: Theclosesttohumans Mammal Themostcomplex Integra>onofsystems(endocrine,immune,nervous,etc.) Gene3cmanipula3onisextremelyversa3le GainofFunc>on(Transgenesis) LossofFunc>on(knockout) ChangeofFunc>on(knockin); Temporallyandspa>allyrestricted(Condi>onal)

MousemodelofHumandisease

HumanvsMouse

Thereareseveralareaswheredierencesbetweenmiceandhumans could be expected to result in divergent disease phenotypes for muta>onsinorthologousgenes: Dierencesinbiochemicalpathways Dierencesindevelopmentpathways Absolute3me Dierencesingene3cbackground

Therecentcomple>onofthemouseandratgenomesequenceshas iden>ed a number of human genes that do not appear to have counterpartsinrodents.

Ethicalimplica3ons
Thoughqulethicaldecisionmakingcannotbeignored Ethicalissuesincludeques3onssuchas: Shouldtherebeuniversalprotocolsfortransgenesis? Shouldsuchprotocolsdemandthatonlythemostpromisingresearchbepermiped? Ishumanwelfaretheonlyconsidera>on?Whataboutthewelfareofotherlifeforms? Shouldscien>stsfocusoninvitrotransgenicmethodsratherthan,orbefore,using liveanimalstoalleviateanimalsuering? Willtransgenicanimalsradicallychangethedirec>onofevolu>on,whichmayresultin dras>cconsequencesfornatureandhumansalike? Should patents be allowed on transgenic animals, which may hamper the free exchangeofscien>cresearch?

Takehomemessages

Plancarefullyyourexperimentaccordingtotheresearchques3on Takeintoaccountthe3merequiredtoobtainallthenecessarysteps. Ifplanningexperimentswithanimals,alwayskeepthenumberofanimalstoa minimum Beawareoftheethicalimplica3onsofyourwork Ifpossible,takeacourseonhowtohandleanimalsforresearchandlearnwith moreexperiencedpeople

Backgroundliterature
Siegel,G.;Agrano,B.;Albers,R.;Fisher,S.;Uhler,M.,BasicNeurochemistry:Molecular, Cellular,andMedicalAspects(1999),Lippincop,Williams&Wilkins,Philadelphia Strachan,T.andRead,A.,HumanGene3cs(1999),GarlandScience,NewYorkandLondon HartwellL.,HoodL.,GoldbergM.,ReynoldsA.,SilverL.,VeresR.,Gene3cs:fromgenesto genomes2ndEdi3on(2004),McGrawHill BruceAlberts,AlexanderJohnson,JulianLewis,Mar>nRa,KeithRoberts,andPeterWalter, MolecularBiologyoftheCell,4thedi3on,(2002),GarlandScience,NewYork