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    Principles of genetic engineering

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    Genetic engineering

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    Principles of genetic engineering

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
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    28 views31 pages

    Genetic Engineering

    Uploaded by

    Pragna Bella
    Principles of genetic engineering

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    of 31
    BEE Pana of Gere mC . i DEG ioeening ? The proces gq alteration 3g maleal by olixect — nbwenkien sin genclee promstes ger te Paoduckon new gene products vv t improve We functions 4 erating ahaamals oF - A Technology 03 enlng a cll & chonging 6 wethig a nmowng Pmt a coud Gondbse Cire) ~ Genebi &n ineering Wes the dechnigos @ molewlar Lerma & hans foumeilion to abr the Shuchita 4 chaatersrtis 7 qe dinectly ; ~ Tromsjer genes bet’ Caganuine thal Unrelated is Tranagnais : ~ Transper o genes ber Ou genims tat are related 4 _Gegenssis — Genekic Enginern can be Wed por @ Emproving up Technology dike producing gee vice, BE cotton © Pooducing apne produc fike Synthelic human Insulin @r7 humolin y Interferon by ming nodfiel bactoia © Monapactare a exython poiekin in hamutter ovary Cell © produc” Expoimental mie — Oncomouse (Canter mouse) for seseanch cli ir ih an in 1973 When Herbert Boyer = Moder aorilic Engirveewnay theg 17a thee Henheek Boye, ard 8k Cohen vad emymes te Gt a Gaclentl plasmid & eed ity another Shand DNA tm he gap _ De lato, Yamilton Smith accidentally pound a DNase oo hacterdirn , Hammophilus inflcenzae that a dua af pabinchome Sittin. 2 Yor. [Gp scanned with Oken Scanner dn lan, Wg, Khorana, an: Inclern oan Kierlil , Heported ‘w total Sypthesta an auhireind deme cued Sth lppie FR he that Can Bunctén within a Living colt g & . _ fn 1977, Cty 9 hope Medical Cuber Duarte, Califor santhesised pre 4 human pwken encoduiy DUA ia jew brole trovmone nom an Somatostatin . & duerted wb dockerca- Phel preducal pestein late ay Irwin gene toed inlo backers & rrcedle. fo Spltere F DNA Technujuas ~ Plasmid and Vector Methods . 2 Biologie! Vectors like plasmids and Views corm gegen re into Colls- — plasmili are Small Circular trtrachromosomel go elemerits that can move bul diff spe and Can replicate by Penelves = Like plasmids, viivea also ads os Veabrein genetic: Enpineeiy Post gq tre Genelec materiel can be auplaced vag: 1 Microlusecnonm Merieps : Method : Iyechn oy gendic maleial new duived gene into Recfpient cell (host) . | For Lage Gt like plant cell 8 animal colle, Injeckim & Lone by fine~ tipped glows nee _ The injected genes get inconpos lad ammeng He host lk genes, | 2. Uedroporation and Chemica) poration method : ~ Divedt ane transper ~ Bovolees Crestitn pores ov holes tr the host Col Membrane. = Poves oWow te enthy Q aia qe Scanned with Oken Scanner CHemical PORATION = Chemica PORATION, a) © Eee trathod © Clb av Immeried in chemise) Solin (cact,) sand Creation 9 pores take place. Blecho povalian + cells or ubjeckd to weak dechric Quant and pores are gored © Bicbatlistics [gene abe pa Wwe metal silvers to oldbier gre materi) to inlerios q Cell - matted, Prjedlile meted. O 2 ® i > ee Heli gas sf coated’ si moll y 7 Dua i Iver. (vey smal) span Cots Soren + ow cs “eo 1 te, sal 33 LS red aw Scream cee Resrejction ENZYMES ; Se NGO ener - Reihicksn enzyme ds an endonucleare which Selectively cleaves the double Stranded ov dingle Skancked Dna at Specigic “recognition nueleobide. Sepance known as srestriction sites . = Resbeiction engymes ore present in baclour ¢ archachaderin, Hare engirmes help & protection. when views Invade any boctesl Call, Ye bacl- Celt mrochiney & wed oy Vr pe repldalion, te veilviclon ergyenes prevent The veplcakan 4 Virol DNA ox phage Dua by Gulbiigs it nb Pieces heme, He name eshnctoon. - the retriction emyres rake tue incisions — once trough eecth, shand ah, Suge photphale backbone q double hole en & Dua. Scanned with Oken Scanner 3 dis coved by Harillan Smith ue L970 ,voa = Fast Resbiction engyne Pig ' awarded a Nobel Pruze- od DL, for whi Me Ome Palincromic Seg uence \ piney pabisne (2 GTAATS) Enverted Repeat Palincome & a@rAarac > \nveted Repeal Palinoleme is mort Common reve & CATA TG 7 Tre ty Compliiuby=G TATAC 1 Bared on Outs produced Reatn dion Enzymes v r 1 | s' = 3 aed | eet uch ard, Mek, | 3) Ender’ 5) é u | L > Cenk. 9 dua als. , Sg) WATIC ; sina : shale sare TTTTs Yaharg Q bass + Ne ovaho piri iives Litt 7s ends | 3! 1 ba 8g st icky - Me Reilvictn &; ! re Neo schizomens Bessa omer a Dige Rent Raalviclion oy. Same Seqpente reegnibin at Ahyernl Socobs = an - ~ Dipy. weshietan enyymes 7 Metegniye Same Seqpitnce 9 ok Same Secdlion. (cor Table a Rettichin Lxslonecltars From bie quien) | [Gp scanned with Oken Scanner jaliure 9 Rbiclam tndlonoclearcs © EB - Gems: Ercheichin Co Speties- cok R Shain - RIND Toten g jt Aarti fied Ielelifp ex” Types OF RESTRICTION ENDONUCEASES oo pocter_vejpuinerens, host DNAy (anathy! trams prone ackohhy) j Type br - Dpol 5. sdS - Speagicthy % seeqribo Type 5S ~ TORT site te be cut. Type Dt — “Bpukor BstT ( Type Hed | Te _ pt vecognds [cat temmen, g mel ved Re, mesos line sepeule cae Pane pe palindacrnic Sequences — cat te oA of oe 4 Ragin sik undivided | now p Seg : chon a ry cane i | =Patindaom’e Seppunce at | va veal P _ Recognition Ste 1 hes 2 porkens \ «yea Nida io Leng co Tg ae | pen nee 3-4 Neds wg neds | | do met ~ pun enelluy alien spacer 4 b-e weds. \ - - ATP - Restriction Invelved in procemes Bike Dna replication, vepaingalamog eo Dna & siecombin ation + > rare ery help in the Jpomalion phosphodieiter, bonds bt ston a adjacent 3! phos phate termi nus « Ligose qe" the nicks. in ds DNA- gone 1 bid —Slivclve 9 pya lige NEN — Dydensfation ~ Se for 07? bid —Conails q fo Brain a dlng) — & tee + WADT cleus ( Compas tva vant OB fold domain. ~ dsdna 7 Dna Canes (boned on woyprremedt & Their fimclury, gee Uses Nagh as Confer usATP 03 conjactor : Faved in probuyet (bachiay ool fy enkarats, ros, bostenphoge = DNA lipase helps in dagaling the nicks onl lange Gps b DvA- =the Con ypttos ATP os Mant Lind te the achive sie 2p Migee rrotecle.. I toa alot bind the DNA Giger Cannot Sevogmige a shah _ wa lire ow Ugeh a gepquple ¢ Nuciohile cheteh, [Gp scanned with Oken Scanner Die Dat ligases ep — temas Nad depended Arp Depencet Jo | J “ a DO Mammalian Baclinnf — Parpelipans v I V : DNA Uigaae 3 Dus lene ae BH Dw Ware QD. 7 vide a 4 ; 2 Ugalis Nested DNA Q > DNA Yepaiy > ain § nwa du: soi a eee ae oie Cobasoei Froqnest) > Cnion- divi ng fcaton 9 "i . > (NIES)- Non- hemologous ees Ce Med one Od ining Pee. >C Velebyatyy Jed | @ Rateril Lave Saad T% DNA Uigares wu AIP ay eneyy Source amd -Can big ake ia plint ends @, Staggeud ends» Temp: aepairel 8 20-25%C. webords ball the Nudeoticle bones ane eanily jovmed . Scanned with Oken Scanner IDNA Peymuone 2 PNA Perron core —— © > Dacoveud by Avthur Koren beng > 1956. pt noun Dus polymerare 2 Helps in potymesisakin Q Nuclestiles « + PA gane Codes jor png polymevane I, Three Frodkdwy! Sosa! [youwaw) dvd polymenare ackvity , template tandl: * 311s" Ceveue) exonvelente achvily mmetiols proprensling - epics Fo pnmn sik and a 5 S48 (fora) exonveleare achvity meduichesy ick tromalation shang, DNA nepady . “Diva Pel T vemoves wa primers created by pamase fom lagging Stand & Bile wth nuclotile b Okagalet progree slat Ad eolidn , ~ Template - elependunt engyme - g adds nvcleclidls Hal — Grweclly base pier [ wilh exabing dvs shard template , ~ Repticabon tats pla at Lovo Nios [sec rate and te sale Q syothesis 4 DNA PealD & 20 Weds] eee . «the dicvey 9 rmutent - Pel by Sohn Gaigns in 1969 prvel teat DNA Pelymeae 2 & pot fhe main empyme Folved tn DUA wpleation. a DNA Polymenre = dnvelved mauindy tn Bepain a DNA -qok da in sie: _ pol 8 gore Codes for DNA Pal D enzyme . | which dondt have BNA Pal J hawe 10 effect in grout ov Scanned with Oken Scanner pid bbs pla in Stalimaay, plane sohenc ~ Supthesis Q PHA ' vt dete 5 resent bey pormnury chest fmm the porielaliiy 4 ‘DNA = Bung Here evenk, DNA Pel I can peinitiak DNA Sprttent yl the ars : ~ has low error tate but & tiv Slow te be a mock we ~ Pol DB doesnot have 59 3) exonocleane actialy & cannot tramnalate the mide - Primary — engyme complex involved in prokaryotic Dua vepbicakiin ; = dibcoveed by Thomas tarenbeng & Malcolm Gefen ip 197 - DNA Pol ML works C Ona PAT, Pl 2, Pol MW. < DNA Pol TH ik a holoenzyme involved in : (© Replication Ackrity © Proof teadung Eee © Component g, Meplisome Docated ot rephealion fork - Replisome & compe Q : 1 we gubvrat with polymenyelion ackvily- gfe & Subveit hos prrh veaduig ackivity - Te 9 Subvet ahiwulstaa the € Subuiue prog reading 2 pons Uamping He DWA pel bound to te templa dua act as gliding DNA clamps - SUT" vals connediog he 2 DMA Pol Bi ergymes. Vr vik = ee fer 2p Uni form | unit do tof it an nd ote DNA. (IP unt & made 0 5 7 subuidy) 3. « in Scanned with Oken Scanner Yenilip Nect0RS (4. sre Vehicles) © Dy “Bi Velon sm Vecton, dan exhachomotemal small ope Mokcle that bar the beak tr am oppoporale frost <, While Containing the desivel wa invest or DNA progment te be Cloned ” = UF Showd fave an ‘Oni’ Site Yor the Ongin 9 Dua replicate tat Can work even in & host all. © Propestia 9 0 Good Vecka_. be Ghoul be able to méplicate aulomomourly in host alle camyiy the asian! gene fragment under nelarel control so Bat if Gr gerrate mmulbple Copia 4 ibd, in a hot all. 2+ Should be eary fo isolate anc Purify * vector. 3. The transformation q the host cel wilh a Vector Should be my: Vector sho be easily intip duced inka hock tells - Eee Vactin should ave Suitable gene marken (selectable mantor genes) that allows easily detection and Selection 03 transformed cell 5, Vetlor should be able t inbegrete ibself inky tte host genome Vector Should have Unigye target files pv the Retlrickion eben Ae aukvily (to ad te ona g duet He desived gee dragnet) Show have minimum amount q nen essential Daa Z 8 Small to Sije Less ham to kbs ia Sige. é ‘ Scanned with Oken Scanner TDN st Dat liane ach the clonihg sits in the plasmid au molliple . ™ ob So mmolhple verticlimn engumes Can Cleave the plaomid, genome many Auris gore fyogmanli can be snbcduced ily te Verdes We Seeetable Marker yens posen he antidiotic nerisank : whieh mate i cay to doket Select the than coll. The | fordhon 4 te antibisfic — gectant poe will not be geen bo te inserted nb the Sthectable markn gh, Bente, the que killed in a Antibnolec medio « The pros Yn Tromsformaliin hw plac: a Freak iprmchidoh : The pram th which the bacteuaf celf Uplaks the ¢/ Lit gor the enioment Called Transpermar whch os [Ney slow Fane we trdice tarspralion ley tration @ dl ach! Pn hp Sof fy -T Scanned with Oken Scanner [ U4. Vectoss clei Wei Rios es: tec payout | 1 Popatee Scone toot jer te reg QA DNA inset whe prwduce veto @ (4 Specigeed prolen. & Called é : : resin Vedo¥ Q BMA meds te eee Foepreaee then ik as Leamel ay Cloning, [ Vectors + Regia - & : } WA 7 ee ewe een =Mulkiple Copies op te Some gene h. Opurators oe prduced. 3+ Ribosomal binding, sikes . (. Tiaracxis & Tranla® pee “lakes place 3 Exyrone a deme) gene maim = fonts 4 dbaied gare & mac AN Veekors (genetic Vehicles) ae wed te Camy daivoble Gens i They lave dipfeut types, dijjenert marker, tify rrestichin sikes, divene yunckions leangabe ard Sukorgobic vectins. They lan be bolm =p” ~ Tramscutp” takes place. “Transcription: Necenof alk Veilors . Proum 4 Synthesis Qn RNA fom Dua . The geralec toformaitden a, DNA Cops) tw RNA. : * mos Scanned with Oken Scanner ° -S F q Promotes ; rp pitake te fomenphon F the compornt necenary fpr all Vectors which 4 used te ay F eoamigiy ay & vol anger Eyprosion Vectin : Neder dsgred fet prilein aa calle, & Pardue 9, ete potuins bs Drtlih ansciphin 7? 19 or ruin, Travsleliro > prban Sethe v arco, A, : Pm 4 prt ad pectin gy news purited do prt hot cots, L goa i Te make te gelechso emry) te cloned gee haul y howe. a bog: j ‘ tag Boul be hitidine (HH) OF rmeake peptide, Scanned with Oken Scanner > Vecde ave meng tomas ¢ ania Vector 3 ©. Phasmisds 7 amb da. d phage . % Phage Vector x 3s Cosmis fe Shollle Wwators J % : 4 Vector . Plaan’ fj 1 Pawn jt cages 9! indypendent > ™P baet seal i 7 not be plont aay ee yr, | 25D © Plovmid vanes fh Sige e vr on Replreation + Then a tuo bps 9 rand «ie Pods © apliati & Did - an Gea ane se Ralewed Plasmas + epixkon & unconblld (mou Cop ey b Fe phamale : Gssporile fr orjgetes) Pe te coyjugaton . Br: F plomid 9 €.cl a. Re 2 Pleamil (genes jor reritance to Anlibiotics), R-plarrmils are Vay wippokank in human health, aie hay male bactunl les Awisbint th: cera Arbbtotes, - Some, “ Avitibiol: yaistamte marker genes used bh qe & R Phoned Plomid Rg Puudomenaa \ | jj i / 4A! te Scanned with Oken Scanner F foonib + Cade for Cobre, he pritaie shih bv @. VJ Semitive oer ae Oot pro Vers, vc ae tet pled, oq a pete CAG{ & wed tr Several vets ay Gb pours plo ampliion 1 Plasmid 2 pee S22 (note gier Abad). ee + These ax genelically ergdveenel Views es : vooditrl Viral DNA ve RNA that non-inieehiovs g contain viral Prmofeu ¢, trangen- = Tromlakon a, Fongere Jo thmugh Wiel promoter. abs vino) Veebos are ran: ingetliins thay asl helper Vinwee Spor. “Trans jeckion : - Virol Yeelou one dusigned Pe perarent tnseiten desired gone fragment ino hat genome. Ex Mis phage , 0 phages Miavivses, dina vis Uke SHO 5 * BPV. (Bovine Rapillomavins ecloss ete. ~ M3 2 } + thy i Hlamentows bachaoptore 9 E cote = Cowinls 9 se Calas dust 9.6408 bp lay: Scanned with Oken Scanner r R ; 2 YF The se pnd -vepteall “ Pe foi} mn ai : b - pil F pilus tn si © = bata nb eho trough FPR. FP on oc vy + adsorption wy Pee ty te cul, Papers § M13 veelie | Adyarilare 013 Veelon: + ge aed con be cloned ; ‘ ‘ i Gr oct a > ds coptes eirwlar genom Dua a Obtanie feat pleamil ; = pra wert can be accepted vk either g the drientabon . =) actesal cell injected by Huse Veclors remain Viable. . Plague Pret Eales pla jor cong selecBion = Rewmbinanl) Dua Gon be Oltasned esithon Stable tactsieplas , particles - ~ bantdrh plage wins particle with head, toi, tail proerne & dahl sharia linear Dud as genele moteral : “oy gage fue DHA sn bot ough Lai genta vn AG,502 bp. Contain Origin g depleatan , genes codsiy ve pote teat ae augpssd te yom ead, tall, 8 y™4 for ta pore og! BO4cqgcqa CCT & Oty end 6004 cg C746 i. 4 as) , pre ies, in eo & Clvtular. " Ceol | He oe lant ™ Coe ey, ile, ‘ A cohesiic ends annens, MW Creal of , en “to plas Scanned with Oken Scanner % 4 ‘ d gt lo & Agtu eee po Clone eo DNA rages / agro » uate ‘dbud Agel PS UST EE ob DNA. sige) lambda phages devo Advantages oy A phage as Vector: - tebe actidhy vets ckion aie ev Resbaction Srolenecbme y ~ They Cortain x ae ~& & provide \miertion stk jor DNA P 9 eI I Belshin q Gene g tnlest ; Qfaihichon Sudo nudeoses OC) Re mut Common mneficd ued fo ticle on4 es by He Use 2 aubicton engymes Or austiction enconurleos eg « iy Clevaye 9 ale mals ut bw Joy aealriclion enya gore Varner eyo Q DNA (y Various fragments are neslved as bands or are Seperate) bared ar Meleewler —werghe ty Agonese gel clechoploress . Ws Specie hands one identifi) by a techwigee Cotled Sorters, blotting « Ger ova yashicbn fragments ale olieehel en and blettel onto niibocetlalove - Se denctinad, O15 the Yl incubated © reioady, ter pe ly lobabl DuA/ana probe Specige wir dabulel probe hydwidise, gy, “ Complement in serhicked pw gba dised pra vabrctal ous & DUE prohe sel a Dw g te e Allo radivgn veal oaling phil, [Gp scanned with Oken Scanner ‘ @ ExMechaniiel Sheanig « 3 rh pager’ ane alee prowl by mechantial thea soles fnkewe sonificalion high wot wl. 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DNA wD Self- replicating entiliy maivthainas ge (pers = Irernttah hie = . —e = oS Lowa Co precipitate } —— en cea eachentehia ult J caith Caleaine phesphele ere oS A vss, polyethylene ih - Bacillus. l= DNA Complered with erandtermiy DNA i ach é oe | diethylario -ehhyl— desion ee entrap DA E Lip oe { alt) then pued with host ed f~ Arofinemy ala Coen - detvany, eat : Membrane) Saccharomy Cais dua trapped) so Uyosomes Tores-tndecs Newretpora ) loonie a . | Eaytterocyte ghosts. Parbadenion plasma, spose Zoning | beeppeeae Priva ceils by “Using Tuse g Liposomes ! 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    _ G&G Ager Contaiiang Seperdd Wei He Membrane and Lacterial Colonies onus te Mow Orginal Gls a Teytreate on tp pil co secover Replica ple on New agen Sin plate» oe ay | pense mrumbrome wilh attached eolonies - Ldeatipy P ye a Sook is Succosie tof Gere 4) Denaturt PE we ee Xray flem by Neutvelige c) wash. Byadigg walle a) yx DNA. cee A MN pao 5a edits Bacteriol Dua Ve ae Cot TD fined om Mehran, [Gp scanned with Oken Scanner ctony Hybiclig alien ® ©@ Tobony Hy dig ation. adie labels DAA Prebe that Comp emi 4 sed * os ured , -On *calons rybiigalen a to athat on pat % the preign / des = the transjormanks , the pacleual Colonies oe allewed te grow neclcom, v Replice plated en te ribocllulese per dre 1 Atlowed te FY OM Subport nobient medium Nilwcellulose a 6 prancl to oltnale PNA by alkali Ireabs hevbraliga” & prlteirare eatin ead te remove prelecins v Filla mubated csth radiolabelled probe v Sivntlenidy 9 ansformed Colonia reembinad DAA with probe, v Pubaye radicgyaoph gee we signal - Raed on Huo, broom formed Colonia are dlatectsd ar master ' ge plate. @Reporter genes + ~ Report, genes ke Lux gene code fpr enzyme olan ot Pat ends gene calf maelation & yor ae Pt o 9 Lamivercort — protecio. - ledfeare Convers Chem - energy inls ial energy cary Luminescence ox glow. “The ges “a incenperatd inte trams }ornasils amokiig tam glow hence Selection cory 8S: q reposto, gens gen Pluorescent gene Hom Telly pb Aeyus rea preducs green bhurreouedt calovies oy tearajormed bacon I [Gp scanned with Oken Scanner © Repro protinn A gal) bac Z ysl Yet & added te He Crllare ML une xg & a modified su : ° Fractuity: ev. te seed He expres eye p-gahcteridare lactose ubilig ction > a genclic frail vs = Tpseslon al activation tate plac in which a gence Ais nw pled by vertices 4 Jove PNA - - alac tesi dare - x- gel -yans blue when hylan! by p 7 e makin oD cls by sel ax an Indias fo inditake the banat plaswil with denied gone pragma. ~ the foreign DNA ocrted divrepts He lacZ gene: Backewal oe acgping the reir Dua poget Ont. Se He forean gen toe pr gulactsidave activity ape tseane ne Bc to Iyholyse he x ga ge : Gite 80 the -hactaval Colonca appear while - Sy ap the actin Clovis dowel tate Me DIA brigmed fost) L : : The Pp qalactasidane empyrs 7° ’ ‘ : in redone This Wpbalyces the x-gal sagan io L The fred with bacterial Colones wl) tuum blue in tee presene a xg. [Gp scanned with Oken Scanner | Ap icobions Gene _Clonivy + © ~ Gene) Cloning, oF recombinant DNA Technolo matted has halos) in tsoltien gene, gtrvetnal analysis Q lange number op Prokaow lis ard Euleanyaec ee ‘hreeagls Molecular Cloning, ~ Bach clone Contains a Single Seapience, oF & pe P ber Vey lange genome + ~ Gene atudtune — funclion nelodiinnalups howe ve intioducin: NA Segpen tes been S$ bedid by Cloned yore ule exdzcnryortee nucle ond dhter tv transcxiption and translation achivihis.. Therapeutic Cloningy : - Gene cloning g a revoubon in Bio-lechnofo ge, 7 / i As io _ scale & posible = Produchio> 2 pelypeptile and priferis adage & poe m cloning » ang " b -A Yoxidty 3 poige pete ean be produced in Ecol. ~ Prcteins aching dike ergy amd pretevia cath ee therparilre applicabons (ke nsulin Interferon » gosh hormones, Vumunogfebabais ets ae produced. ~ Dally, the lamb 02s edd by tepudlae -clomagp technology MY Reproductive cloning & Technology vse te gunerwile on arima thet has the Some ywelean DNA as another Currzetly exuling animal oo whieh enisted prewtounly - ~ Dolly wos genoa by © pram © which the artis materiel from the mules Q & donor adult ctl is transfered fe an enveleated wahose ze ométeiad or nudeus i, vemered “48 Scanned with Oken Scanner is weconstvuctd , ama having PNA from donor cet wt with chemial or electric Current 80 that cell olivasion ue ; Com Stark / cloned embyo Stable and Teaches « shibable : re ‘ then ‘Cransfered to the Uberws 04 Jemale host, mit then it Gorilinves ty develop vynt{ bith.

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