Cytochrome P450
humanised
mice
F
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k J
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G
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Laboratory of Metabolism, NationalCancerInstitute, Building37, Room 3106B, Bethesda, MD20892, USA
Correspondenceto:Tel:þ1 301496 9067;Fax:þ1 3014968419;E-mail: [email protected]
Datereceived(in revised form):26th March2004
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Humansare exposedtocountlessforeigncompounds,typically referredtoas xenobiotics.These caninclude clinically used drugs, environmental pollutants, food additives,pesticides, herbicidesand even natural plantcompounds.Xenobioticsaremetabolisedprimarilyin theliver, butalsoin the gutandother organs,toderivatives thataremore easilyeliminated from the body.In some cases, however, a compound isconvertedtoanelectrophilethatcancause cell toxicityandtransformation leadingtocancer.Amongthemostimportant xenobiotic-metabolising enzymesarethe cytochromesP450 (P450s).These enzymes representasuperfamily ofmultiple forms that
exhibit markedspeciesdifferencesin theirexpressionand catalytic activities.To predicthowhumans will metabolisexenobiotics, including drugs, human liverextractsandrecombinantP450shave been used.Newhumanisedmouse modelsare being developedwhichwillbe
of great value in thestudy of drugmetabolism,pharmacokineticsandpharmacodynamicsin vivo, and incarryingouthuman risk assessment ofxenobiotics.Humanisedmice expressing CYP2D6and CYP3A4,two majordrug-metabolising P450s, haverevealedthe feasibility ofthis
approach.
Keywords:humanisedmice, cytochromesP450,pharmacokinetics, bacterialartificialchromosome,transgenicmice
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CytochromesP450 (P450s)and other foreigncompound-or xenobiotic-metabolising enzymesaretheprimary interface between the chemicalenvironmentandthe body.Xenobiotics include drugs, environmentalcontaminantsand food additives. Theycan also encompass natural plant chemicals such as flavonoids,steroidsandphytoalexins.Sincethere are countless differentcompounds thatcanenteran organism,theremustbe ameans for theirelimination.Thisis particularlyimportant forhydrophobic chemicals,which candissolve in lipidsand accumulate in the body,or thosethat can react with cellular macromoleculesand causetoxicity and cancer.In order to copewiththeonslaught of chemicalinsults,mammalshave evolved alargenumber of enzymes thatappear tofunctionin the metabolism of xenobiotics. Althoughsomemetabolism of endogenouschemicalshasbeenfound, thephysiological relevanceofthesereactionsis unknown.Asa general par a-digm,xenobiotic-metabolising enzymesconvert lipid-soluble foreignchemicalsintoderivatives thatcaneasilybe eliminated from the body.
Xenobiotic-metabolising enzymeshave historically been grouped into twofunctionallydistinctclasses,thephase1and phase2 enzymes (Figure1).Phase1enzymesincludethe P450s, flavin-containing monooxygenases (FMOs) and epoxide hydrolases (EH). The phase1 enzymes use O2 and
reduced nicotinamide adenine dinucleotidephosphate in
‘oxidation’ reactions.Thephase2 enzymes,which
include the glutathione S-transferases (GSTs),uridine dipho -sphate-glucuronosyltransferases (UGTs),N-acetyltransferases (NATs),sulfotransferases (SULTs),methyltransferasesand a few others, add hydrophilic derivatives tocompounds to make them morewater soluble andmore easilyeliminated from the body.These conjugation reactions usuallyinvolveoxygen (hydroxyl orepoxide groups), nitrogenand sulphuratoms. Thus,xenobiotic-metabolising enzymesareresponsible in largepartfor the disposal of foreignchemicalsfrom the body. In the caseof clinically used drugs, xenobiotic-metabolising enzymesgenerally metabolise and inactivate drugs;in the absenceofmetabolism,manydrugs would stayin the body and remainbiologicallyactive for longperiods oftime, a property that would, in most cases, be unfavourable and lead to prolonged drug action.In some cases,prodrugsare con -vertedtoactivemetabolitesby xenobiotic-metabolising enzymes.A list ofxenobiotic-metabolising enzymesis shown inTable1.
inactivatedwhileothersare activatedto electrophilic derivativesby xenobiotic metabolism.
The ability of xenobiotic-metabolising enzymes tohandle manydifferent types of chemicalsisdueto the fact thatasingle enzyme can metabolise alargenumber of different substrates. The substrate-binding site from asingle P450canaccommo -date differentchemical structures.Oxidationcan occurat different sites on the samemolecule. There isalsoa high degreeof overlappingsubstratespecificitiesamongthese enzymes,wherebyasingle compound canbemetabolised by
severalenzymes.Thus, alimited number of P450sare ableto metabolisescores of different chemicals, and thiscould accountfor the tremendouscapacityfor mammals with a limitednumber of enzymes to metabolisemanydifferent xenobiotics.
The xenobiotic-metabolising enzymesare important determinantsfor boththe favourable(disease or symptom ameliorationinduced by drug action) and unfavourable (toxicity and carcinogenicity) responses to foreignchemicals. Patientscandiffer in both their responses to drugs and their susceptibility to chemically-induced toxicity dueto the large degree of inter-individual differencesin levels of expression. Since these enzymesactivate carcinogens,there could be a difference in susceptibility tocancer which depends on levels of xenobiotic-metabolising enzymes. Thelatterhasbeen examined by case control epidemiology studies that have attempted to determinethe associationbetweencancer and the presence ofmutant allelesfor xenobiotic-metabolising enzymes.9,10
There aremarkedspeciesdifferencesin the expressionand catalytic activities ofxenobiotic-metabolising enzymes. Asa result,rodentsand humans can metabolise asingle chemical entity quite differently.Infact, even ratsandmice,which have beenevolutionarily separated byat least 17 million years, have uniquesets ofxenobiotic-metabolising enzymes. In particular, the xenobiotic-metabolising P450s show a high degreeof speciesdifferencesin thenumber of genes,regulation of genes andsubstratespecificities of individualenzymes.11Inhumans,
there are alimitednumber of P450s that are known to metabolise clinically used drugs, andthese enzymesexhibit substratespecificitiesandregulatory patterns thatcan markedly differfrom P450sin mice andrats.Infact, cloning and sequencingof P450sand, morerecently,totalgenome Figure1.Scheme depictingphase1andphase2drug
metabolism.R1and R2can representanyaliphaticoraromatic
substituent.SGrepresents theresults of a conjugation reaction
byglutathione S-transferase.
Table1. List ofxenobioticmetabolising enzymes
Enzymes Reference
Phase1
Cytochrome P450s (P450 orCYP) http://drnelson.utmem.edu/CytochromeP450.html
Flavin-containingmonooxygenases (FMO) 1
Epoxide hydrolases (mEH,sEH) 2
Phase2 ‘transferases’
Sulfotransferases (SULT) 3
UDP-glucuronosyltransferases (UGT) 4
Glutathione S-transferases (GST) 5
NAD(P)H-quinoneoxidoreductase(NQO) 6 N-acetyltransferases (NAT) 7 mEH andsEH aremicrosomalandsoluble epoxide hydrolase.
Abbreviations: UDP¼uridine diphosphate;NAD(P)H¼reducednicotinamide adenine dinucleotide(phosphate).
sequencing haverevealed102 putativelyfunctionalgenesand 88pseudogenesin the mouse and 57 putativelyfunctional genesand 58pseudogenesin the human.12Thus, humanshave
fewerfunctionalP450sgenes than mice, aninteresting fact that mayhelp todevelopa hypothesis on the driving forces ofthe evolution ofxenobiotic-metabolising enzymes.11The human
P450salsoexistinanumber of allelic forms, and in some cases wherethere aremutantgene-inactivatingmutations,theselead toP450 polymorphisms (http://www.imm.ki.se/CYPalleles). ThemajorP450sinvolved indrugmetabolismare CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.Those P450s thataremainly involved in themetabolism of toxicantsand carcinogens are CYP1A1, CYP1A2, CYP1B1, CYP2A6and CYP2E1.Themajor polymorphic P450sare CYP2A6, CYP2C9, CYP2D6and CYP2C19.OtherP450sexhibit marked inter-individualdifferencesin levels of expression which arenot dueto polymorphisms,notablyCYP3A4.13
Since drugsaremetabolised and inactivated byP450s,these enzymesdictatetherateof drug elimination.In manycases, a patient must takemorethan onemedication, andthiscan result indrug interactionsif both agentsaremetabolised by thesame P450.This phenomenon,referredtoas ‘drug interactions’, can leadto toxicityduetoinhibition ofmetabolismandthe resultanthigh andsustainedserum levels ofoneofthe drugs. Thus, itiscritical todeterminewhich P450is responsible for metabolising aparticulardrug.Infact, itis now routinepractice for pharmaceuticalcompanies toestablishwhich P450s metabolise a drug before itis subjectedtoclinical trials.
Over thepast ten years, ithasbecomestandardpracticeto determine how a drug candidate is metabolised and which P450form is responsible forits metabolism.Thisaidsin the prediction of drugsafety.In particular, byknowing howa drug is metabolised, it is possibletodeterminewhether therewill be drug interactions and whether theremay beproblems with inter-individualdifferencesin metabolismand clearance caused by P450 polymorphisms.The best scenariofora drug companyis todevelopa drugthat is metabolised by several forms of P450.Todetermine howa drug candidate is metabolised, human liver slices, human liverhepatocytesand human liver microsomescanbeused inconjunction with inhibitors of specific P450 formsand antibodies to specific P450s.14 Recombinant humanP450s, expressed in systems such asbaculovirus, have alsogainedwidespreaduse.15,16
In practice, both human liver-derived cellsand extractsand recombinantP450sareusedtodetermine howa compound is metabolised.High-throughput roboticscreening procedures have beendevelopedusing recombinant P450sin order to predict howdrug candidates will bemetabolised inhumans. Todeterminewhich P450 has the highest rateofmetabolism foraparticular compound, standard Michaelis–Menton kineticsareusedtoestimatethe relativerateof clearanceor Km/Vmax.Inaddition, it should alsobenotedthatcomputer modelling hasbeen of somevalue in predictingmetabolism with certainhumanP450s,such asCYP2C9and CYP2D6.17
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Two strategiescanbeusedto produce a humanisedmouse. Historically,placingthe cDNA behind atissue-specific promoterhasbeen usedto maketransgenicmice by standard pronuclei injection ofrecombinantDNA.Forexample,therat albumin or mousetransthyretin promotercanbeusedto make transgenicmicethat would allowfor tissue-specific expression in liver.Asanexample,the humanfoetalCYP3A7was expressed in liver using ametallothionein-1 promoter.18
Asecondstrategyfor making humanisedmice is to use genomic clonescontainingthe complete geneupstream sequences that include allcis-actingregulatoryelements.Forexample, a bac-terialartificialchromosome(BAC)clone canbesuccessfully usedtogenerate atransgenicmouse.This would allowfor tissue-specific and inducibleregulation ofthetransgene andthus is thepreferredmethod for making a humanisedmousethat would bethemostbiologically predictive.The geneof choice canbe foundon the Celeraor public humangenesequence databases, and asuitable BAC clone containingthe fullgene and its regulatoryelementsidentified andused asatransgene. The BAC clone containingthe fullgeneshouldnotcontain othergeneopen reading frames.Several transgenic founders (independent offspring derived from the egg injections) should be characterisedtofind alinethatexhibitsexpression ofthetransgene inatissue-specificmanner that reflects the expressionfound inhumans.In the experienceofthe author, BACtransgenesarestable and expressedover manygenerations.
CYP
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Mice contain nineCyp2Dgenes;however,noneofthese genesappear toencode P450s with catalytic activities similar toCYP2D6,theonly CYP2D geneproduct expressed in humans.12,19In the absenceofthisactivity, humanisedmice
canbe developed byinsertion oftheCYP2D6gene into the wild-typemouse genome. ACYP2D6-humanisedmouse was producedusing alambdaphage genomic clone containing the wild-typeCYP2D6gene.20 Interestingly,severalfounder
lines wereproduced, allexpressing CYP2D6 proteinin liver. A coupleoftheselinesalsoexpressedthe enzyme inkidney, however, andone expressed CYP2D6in theliver, kidneyand smallintestine — known sites of expression of thisP450in humans.21 This latter linewas usedto determinethe activity
of CYP2D6 toward debrisoquine, anantihypertensive bet a-adrenoceptorblocking drugthat is metabolised byCYP2D6 primarily through 4-hydroxylation.This reactionhasbeen used for many yearsasanin vivoandin vitrometabolic probe forCYP2D6,sincethe discovery in 1977 ofthe CYP2D6 polymorphism.22The humanisedmicewere abletoefficiently
metabolise debrisoquine,withpharmacokineticparameters and urinary metabolitelevels reflectiveof humanextensive metabolisers of debrisoquine.21Toanalysethemetabolism
carriedout. Mice are administered debrisoquine by oral gavage and bloodsamples takenat various timesandsubjected toanalysisby liquid chromatography-mass spectrometry/mass spectrometry.Thesestudies revealed that mice having two copies ofthe transgene clusterhad a lowarea under the curve(AUC)fordebrisoquine and a high AUC for 4-hydroxydebrisoquine,whilewild-typemiceproducedlittleof themetabolite and had a high AUC for theparentcompound (Figure2).Mice hemizygousfor thetransgene cluster were intermediate between wild-type and homozygote.
Asecondmeasureof debrisoquinemetabolismin vivois determination ofthemetabolicratio (MR) of debrisoquine/ 4-hydroxydebrisoquine in theurine,thestandardmethod for analysing debrisoquinemetabolisminhumans.Thisanalysis revealedthat the MR in wild-typemicewasaround10and that of CYP2D6-humanisedmice, both homozygousand hemizygous,wasabout 1; thelatter value is similar to that found inhumanextensivemetabolisers.Thesestudiesconfirm that wild-typemice aresimilar tohuman poor metabolisers of debrisoquine, andthatCYP2D6-humanisedmice aresimilar toextensivemetabolisers.Thesestudiesestablishthe feasibilityof using humangenomic clonesfor theproduction of P450 -humanisedmicethatexhibitin vivometabolic activity thatis
similar tohumans.Humanisedmice have alsobeen usedto determinethemechanism oftissue-specific expression of CYP2D6.TheCYP2D6transgenewasbred intoaliver-specific null mouse forhepatocyte-enrichednuclearfactor4alpha (HNF4alpha).23Micelacking expression of HNF4alpha in the
liverhavemarkedlydecreased expression oftheCYP2D6 transgene, indicating arole for thisfactorin the control ofthe liver-specific expression of CYP2D6 (Figure3).Expressionis not lostin the absenceof HNF4alpha, however, indicatingthat other liver-enriched factors mayhave arole initsexpression.
CYP2D6 metabolisesalargenumber of drugs thatare used in thetreatment ofpsychiatric disorders, includingthe antidepressantsamitriptyline, fluoxetine and imipramine, and the neuroleptic agentshaloperidoland risperidone.In addition, CYP2D6 carries out the demethylation of codeine to themorepotent analgesicmorphine.These datasuggested that endogenous neurochemical substrates mayexistfor CYP2D6.Molecular modelling hasbeen used toanalyse CYP2D6 substrates.This revealedthat 5-methoxytryptamine (5-MT)isapotential substrate for thisP450. Indeed, 5-MT wasfound tobe demethylated to5-hydroxytryptamine (5-HT), alsocalledserotonin, by recombinantCYP2D6and transgenic CYP2D6.24 Conversion of 5-MT to5-HToccurs
Figure2.Thepharmacokinetics of debrisoquine inCYP2D6-humanisedmice.Time courseofserumconcentrations of(A)debriso
-quine(DEB)and(B)4-hydroxydebrisoquine(4-OH-DEB)from wild-type, CYP2D6-humanised heterozygousand CYP2D6-humanised homozygous mice after onesingleoraladministration of DEB(2.5mg/kg).Venousbloodwas obtained0,0.5,1,2, 4,6, 8,12and24 hoursafterDEB administration.Values represent themeanandthevertical lines thestandard errors ofthemean of DEB and 4-OH-DEB from threemice.
ata higher rate in the CYP2D6-humanisedmice ascompared withthewild-typemice.Thesestudies revealed anew role for CYP2D6 in the serotonin–melatonincycle.
CYP
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CYP3A4 is themostimportant ofthe humanP450s for the metabolism of drugs.It is themost abundantlyexpressed P450inhuman liver microsomesand isknown to metabolise morethan 60 percent of all therapeutic drugs used in the treatment ofmanydisorders, including hypercholesterolaemia (statindrugs), bacterialinfections (erythromycin) and
autoimmunity (cyclosporine). Thus,thepotentialfordrug interactionsisgreatand is of concernfor the development of newdrugs.25Inhumans, fourCYP3A P450sare expressed:
CYP3A4, CYP3A5, CYP3A7and CYP3A43. Interestingly, mice have eightCYP3Agenes.12The mostabundantly
expressedCYP3A gene inhumans isCYP3A4.ThisP450is especially of interestbecause itis not onlyexpressed in liver, butalsoin the gut,where itcan metabolise alargenumber of orally administered drugs.
TogeneratemicethatexpresshumanCYP3A4, a BACwas used asatransgene.26 Thesemice expressed highlevels of
CYP3A4protein in thesmallintestine.Surprisingly, little expression wasfound in the liver, amajor siteof CYP3A4 expressioninhumans;however,recent studieshaverevealed constitutive and inducible expressionin thelivers of female mice(Yuetal.,unpublishedresults). Toevaluate CYP3A4 catalytic activity,the drugmidazolam was used, since itisa standardprobe forCYP3A4 activityinhumans.27Midazolam
is oxidised at the 4 and 10positions byCYP3A4.The phar
-macokinetics ofmidazolam metabolism were determined upon oraladministration ofthe drug.DifferencesinAUC for theparentalcompound andtheprimary 10-hydroxymidazolam
were detected between the CYP3A4-humanised and wil d-typemice, indicatingthat thetrangenicshave a higher rateof midazolam metabolismand clearance(Figure 4). Nodiffer -ences wereobserved between thetransgenic mice and wil d-typemicewhen the drugwasadministered intravenously. Thesemicewill aid in thein vivoanalysis oforallyadminis -tered drugs that aresubstratesforCYP3A4.They willalsobe ofvalue indeterminingthe mechanism of tissue-specific and inducibleregulation of the CYP3A4gene.
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Xenobiotic-metabolising enzymesarethe interface between humansandtheirchemicalenvironment.Whiletheyare absolutely required for the elimination of clinically used drugs, theycan leadtoadverse drug effectsduetodrug interactions andpolymorphisms.These enzymesare also responsible for the activity of chemicalcarcinogens.Thus, drugsand chemicals to beused inhumans mustcarefullybetestedtodetermine how they willbemetabolised.Whilein vitrosystemshave been developedto predicthuman metabolism of drugs, analysis of drugmetabolismin vivo,usingrodents, can leadto uncertainty dueto speciesdifferencesin xenobiotic-metabolising enzymes. Humanisedmice expressing humanP450scanbeusedtofill the gapbetweenin vitroandin vivotestingsystems.The CYP2D6 -and CYP3A4-humanisedmousemodels validatetheproduction anduseofthesenew modelsfor thestudy ofin vivopharmaco -genetics.The challengeremains, however,to produce fully humanisedmice in whichthe endogenous mouse genesare knockedout.Whiletargeted gene disruptionhasbeen usedto knockout theCYP1familyandCYP2E1,most oftheCYP2 subfamiliesin mice consist ofmultiple genes.Forexample,the
CYP2andCYP3Aloci in mice consist ofnine and eightgenes, respectively,spanning hundreds of kilobases of DNA.Thus, standard gene knockout technologycannotbeusedtodelete thewholesubfamily.While each gene canbe disrupted, Figure3.Regulation oftheCYP2D6transgene byHNF4alpha.
The figureshowsa Northernblotanalysis ofmouseliver
HNF4alpha expression.Hepatic RNAs (10 mcg)from
HNF4alphawild-type:CYP2D6þ/2 (lanes 1,2);HNF4alpha
liver-null:CYP2D6þ/2 (lanes 3, 4);HNF4alphawild-typ e:-CYP2D62/2(lanes5,6);and HNF4alphaliver-null:CYP2D6 2/2 (lanes 7, 8) wereseparatedona1 percentagarose gel,
transferredtoanylon membrane and hybridisedwith an
HNF4alpha exons4 and 5 cDNAprobe.HNF4alphawas
completelydeleted afterCre-mediatedrecombination.Bet a-actin was used asaloading control.Westernblotanalysis of CYP2D6 proteinexpressionin liver microsomal protein (40 mcg)derived from thesame groups ofmice as thoseused forRNA analysis.Deletion of HNF4alpharesulted inaloss of
themodified genescan neverbe joinedonasingle chromosome dueto their proximities.Nevertheless,the Cre-loxPstrategyisa methodthatcanallowalargesegment of DNA containing thewhole P450 subfamily tobe deleted.28LoxPsitesare inserted
ateach endofthe P450 locusbyconsecutivetransfection of
recombinantDNAsintoembryonicstem (ES)cells usingstan -dardprotocols (Figure 5).29The interveningsegment of DNA
canbe deleted inES cells usingtransfected Crerecombinase. Alternatively, deletioncanbe carriedoutin themouse embryo oradult mouseusing a Crerecombinasetransgene.By useofthis method, all ofthenon-essential xenobiotic-metabolising P450s canbe deleted andreplacedwiththeirhumancounterparts, leadingtoa fullyP450-humanisedmouse.
q United StatesFederalGovernment
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-trations of(A) midazolamand(B) 10-hydroxymidazolam (10-OH-MDZ)from wild-type(WT), CYP3A4-humanisedmice(Tg-CYP3A4) after onesingleoraladministration ofmidazolam.Venousbloodwas obtained0,0.5,1,2, 4,6, 8,12and24 hoursafteradministration.
Values represent themeanandthevertical lines thestandard errors ofthemean ofmidazolamand10-OH-MDZ from threemice. Notethe higher maximalconcentration (Cmax)for midazolaminWTmice comparedwith Tg-CYP3A4mice.
Figure 5.Useofthe Cre-loxPstrategy todelete anentire
subfamily of P450genes.The individualgenesare denoted by rectanglesandtheloxPsitesby triangles.Thesequenceofthe
locuscanbeobtained from thepublicorCelera database, and bacterialartificialchromosome clones thatflankthe complete cluster of genescanbe chosen.LoxPsitesare introduced by useofin vivorecombination togenerate constructsfor transfectionintoembryonicstem (ES)cells.29Thisiscarried
outby twoconsecutivetransfectionsintoES cellsfollowed by treatment with Crerecombinasetodeletethe DNA between theloxPsites.The ES cellscan thenbeusedto make a knockout mouse.
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