I nduci bl egenet ar get i ng ( TheCr el oxsy st em) I tI ti samet hodofgenet ar get i ngt hatal l owst hei nduci bl ei nact i v at i onofat ar get gene.I twaspr esent edi nmi ce. I nduci bl egeneexpr essi onsy st emsar ef av or edov erst abl eexpr essi onsy st emsi na wi dev ar i et yofbasi candappl i edr esear char eas, i ncl udi ngf unct i onal genomi cs, gene t her apy , t i ssueengi neer i ng, bi ophar maceut i cal pr ot ei npr oduct i onanddr ugdi scov er y . Thi si sbecauset heyar emost l yr ev er si bl eandt husmor ef l exi bl et ouse.Fur t her mor e, compar edt oconst i t ut i v eexpr essi on, t heygener al l yexhi bi tahi gheref f i ci encyandhav e f ewersi deef f ect s, suchascel l deat handdel ay edgr owt hordev el opment .Empower ed bydecadesofdev el opmentofi nduci bl egeneexpr essi onsy st ems, r esear cher scannow ef f i ci ent l yact i v at eorsuppr essanygene, t empor ar i l yandquant i t i v el yatwi l l , dependi ng onexper i ment al r equi r ement sanddesi gns. Thef actt hatsomel et hal i t y causi nggenesoressent i al genesar ei mpossi bl et o ov er expr essorknockout , f i net uni ngt hei rexpr essi oni snecessar yf ort heanal y si sof t hei rf unct i on.Mor eov er , t hei r r ev er si bl emani pul at i onofgeneexpr essi onof t endr i v es compensat or yadapt at i oni nhi gheror gani sms.Ther ef or e, t heabi l i t yt oswi t cht hegene expr essi ononandof fort omodul at et hel ev el ofgeneexpr essi oni naquant i t at i v eand t empor al waycanpr ef er ent i al l yr ev eal t hedi r ectconsequenceofacer t ai ngenet i c changeandpr ov i deanaddi t i onal f i l t ert oexcl udeot hersi de-andof f t ar getef f ect s.Thi s i sespeci al l ybenef i ci al whenwor ki ngwi t hmammal i ancel l st hatar emai nt ai nedand cont r ol l edbyhi ghl yi nt r i cat egenet i cnet wor ks. Themet hodusesani nt er f er onr esponsi v epr omot ert ocont r ol t heexpr essi onofCr e r ecombi nase.Her e, Cr ewasusedt odel et easegmentoft heDNApol y mer ase[ bet a] genef l ankedbyl oxPr ecombi naser ecogni t i onsi t es.Del et i onwascompl et ei nl i v erand near l ycompl et ei nl y mphocy t eswi t hi naf ewday s, wher easpar t i al del et i onwas obt ai nedi not hert i ssues.Thi smet hodcanbeusedf ort hei nduci bl ei nact i v at i onofany ot hergenei nv i v o. TheuseofCr e/ l oxPr ecombi nat i onsy st em i ngenet ar get i ng Cr er ecombi nasei s a38kDapr ot ei n f r om t he bact er i ophageP1 t hatmedi at es i nt r amol ecul arand i nt er mol ecul arsi t especi f i c r ecombi nat i on bet weent wol oxP si t es( l ocusofXov erofP1) .Thel oxPsequencei s34bpl ongandconsi st soft wo13bp i nv er t edr epeat ssepar at edbyan8bpnonpal i ndr omi c( asy mmet r i c) sequencewhi chdi ct at est heor i ent at i onoft heov er al l l oxPsi t e.Twol oxP sequencesi nopposi t eor i ent at i onmedi at et hei nv er si onoft hei nt er v eni ng DNAbyCr er ecombi naser at hert hant hanexci si onwhi l et wosi t esi nt he sameor i ent at i onmedi at eexci si onoft hei nt er v eni ngDNAbet weent he si t esaf t erwhi chonl yonel ox Psi t er emai ns.I ft hel oxPsi t esar el ocat edon di f f er entchr omosomes, Cr er ecombi nasecanmedi at eachr omosomal t r ansl ocat i on ( A)Si ngl el oxPsi t et hatcont ai nst woi nv er t ed13bpr epeat s, separ at edbyan asy mmet r i c8bpl ongsequence.Thet y peofCr emedi at edr ecombi nat i oni sdependent ont heor i ent at i onandl ocat i onoft hel oxPsi t es. ( B)Cr eexci sesaci r cul armol ecul ef r om bet weent wol oxPsi t espl acedi nt hesame or i ent at i on. ( C)Cr ei nv er t st heDNAsequencebet weent wol oxPsi t esposi t i onedi nopposi t e or i ent at i on. ( D)Cr emedi at edr ecombi nat i onbet weent wodi f f er entl i nearDNAmol ecul es( e. g. chr omosomes) , eachcont ai ni ngal oxPsi t e, r esul t i ngi nt heexchangeoft heDNA r egi onsf l anki ngt hel oxPsi t es. Condi t i onalgenet ar get i ngusi ngt heCr e/ l oxPr ecombi nat i onsy st em : TheCr e/ l oxPsy st em hasemer gedasausef ul t ool i ngenet i cmani pul at i ons( 86) .Gener al l y , any DNAsequenceofi nt er estcanbedel et edbyf l anki ngi twi t hl oxPsi t es.Cr e/ l oxPenabl es, f or exampl e, t hedel et i onoft hesel ect i onmar keraf t ersuccessf ul i nt egr at i onoft het ar get i ngv ect or i nt ot hegenomeofEScel l sormi ce.Thel oxPsi t escanbei nt r oducedi nt ot hegenomi cl ocusof i nt er estbyhomol ogousr ecombi nat i onasdescr i bedabov e.Fur t her mor e, t hecondi t i onal ( t i mel y orspat i al l ycont r ol l ed)expr essi onofCr er ecombi naseenabl est odet er mi ne, wher e( e. g.i n whi chcel l t y peort i ssue)andwhen( atwhi cht i meofmouse’ sl i f eorofdev el opment al st ageof cel l s/ t i ssues)t hedel et i onoft hef l oxedDNAsequenceshoul doccur .Thus, f orcondi t i onal si t especi f i cgenomemodi f i cat i on, t womousel i nesar eusual l yneeded. Fi r st , mi cet hathav eDNAsequenceofi nt er estf l ankedbyl oxPsi t es( f l oxed) . Second, Cr er ecombi naset r ansgeni cmi ce, i nwhi chCr ei sexpr essedundert hecont r ol ofapr omot ert hati sact i v ei nspeci f i ccel l t y pesort i ssues, orCr ei st r ansi ent l y expr essedundert hecont r ol ofapr omot ert hati sact i v eatapar t i cul ardev el opment al st ageoft i ssuesorcel l s. Whencr ossi ngt hef l oxedmousewi t haCr et r ansgeni cmouse, t hef l oxedDNAsequencei s subsequent l ydel et edi nt hecel l t y pesort i ssues, wher eCr ei sexpr essed.Col l ect i ondat abases ofsev er al hundr edsofCr et r ansgeni cmousel i nesexpr essi ngt heCr er ecombi nasei nspeci f i c t i ssuesorcel l sar eav ai l abl e. TheCr e/ l oxPsy st em enabl esnotonl yt hedel et i on( shutof f )ofgenesofi nt er est , buthasbeen pr ov edt obesuccessf ulal sot ospeci f i cal l yt ur non( act i v at e)t heexpr essi onofanygene( or t r ansgene)ofi nt er est . Fort hi s, t hegeneofi nt er esti sr ender edqui escente. g.byi nt er posi ngf l oxedpol y adeny l at i on si gnal sequencesmedi at i ngpr emat ur et r anscr i pt i onar r estwi t hi n.Af t eri nt er cr ossi ngwi t haCr e t r ansgeni cmouseordel i v er yofCr ei nt of l oxedt r ansgeni cmi ceusi nge. g.Cr eexpr essi ng adenov i r uses, t hef l oxedpol y adeny l at i onsi gnal sequencescanber emov edbyCr emedi at ed exci si on, r esul t i ngi nt heact i v at i onoft hegeneexpr essi oni naspeci f i ccel l t y peort i ssue. Si gni f i canceofCr eLoxsy st em — SuchCr e/ l oxPmedi at edgeneact i v at i onhasbeenausef ul appr oacht oav oi dhar mf ul ef f ect sof t het r ansgenedur i ngmouseembr y ogenesi s, ort hei nduct i onofi mmunet ol er anceagai nstt he t r ansgenepr oduct , f orexampl ei nt hecaseofv i r al genes.Fori nst ance, t heappl i cat i onoft he Cr e/ l oxPt echnol ogyhasenabl edt ogener at et r ansgeni cmi cet hatcondi t i onal l yexpr esshuman hepat i t i sCv i r ust r ansgenesuponi nt r av enousadmi ni st r at i onofCr eexpr essi ngadenov i r us, and t husenabl edt hei nv est i gat i onoft hei mmuner esponsest oandpat hogenesi sofHCVi nf ect i on.
