Informacija

10.3: Plazmidi se lako izoliraju iz bakterijskih stanica - Biologija

10.3: Plazmidi se lako izoliraju iz bakterijskih stanica - Biologija



We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Izolacija plazmida koristi jedinstvena strukturna svojstva plazmida. Bez obzira na postupak izolacije, opći principi izolacije plazmida su isti.

Slika i odlomci na suprotnoj stranici sažimaju korake i opće principe koji se koriste za izolaciju plazmida.

  1. Liza i denaturacija - Jaki uvjeti denaturacije koriste se za slabljenje žilave bakterijske stanične stjenke. Najčešći postupci koriste kombinaciju jake baze i deterdženta. Deterdženti pomažu pri otapanju lipida u staničnoj stjenci, dopuštajući denaturantima da uđu u stanicu. Proteini su zbog svoje krhke strukture nepovratno denaturirani. Tretman također razbija vodikove veze koje drže zajedno dvije niti DNK spirale.
  2. Neutralizacija - Neutralizacija omogućuje komplementarnim lancima DNA da se ponovno ozdrave i uzrokuje taloženje proteina. Plazmidi se renaturiraju jer imaju super namotane strukture koje su držale dva lanca spirale zajedno tijekom denaturacije. Kromosomska DNK se, međutim, ne može obnoviti jer su se njeni duži lanci pomiješali s denaturiranim proteinima. Uzorci se moraju lagano miješati u ovom koraku kako bi se spriječilo fragmentiranje duge, kromosomske DNA na komade koji bi se mogli ponovno žariti i ko-pročistiti s plazmidima.
  3. Centrifugiranje - Plazmidna DNA odvaja se centrifugiranjem od velikih agregata istaloženih proteina i kromosomske DNA.
  4. Dodatno pročišćavanje - Plazmidi se dalje pročišćavaju organskom ekstrakcijom ili adsorpcijom u smolu.

Plazmidi s kojima radimo u ovom laboratoriju održavani su u laboratorijskom soju Escherichia coli. E coli je proteobakterija koja normalno nastanjuje crijevni trakt toplokrvnih sisavaca. Virulentni sojevi E coli koji se pojavljuju u vijestima stekli su, često lateralnim prijenosom gena, otoke patogenosti koji sadrže gene za faktore virulencije, toksine i adhezijske proteine ​​važne za invaziju tkiva. Otoci patogenosti nisu prisutni u laboratorijskim sojevima, koji su derivati E coli soj K12. K12 je oslabljeni soj koji nije u stanju kolonizirati ljudsko crijevo. Razmnožava se i sigurno koristi u laboratoriju više od 70 godina. The E coli sojevi korišteni u molekularnoj biologiji također su odabrani da toleriraju veliki broj plazmida.

Koristit ćemo komplet ZyppyTM (Zymo Research) za pročišćavanje plazmida od transformiranihE. coli naprezanja. Završni korak pročišćavanja u postupku uključuje spin kolonu od silicijeve smole. Nukleinske kiseline čvrsto se vežu za silicijev dioksid u prisutnosti visokih koncentracija soli. Nakon koraka ispiranja kojim se uklanjaju preostali proteini, plazmidi se eluiraju otopinom s malo soli. Otopine plazmida su vrlo stabilne i mogu se dugo čuvati u hladnjaku ili zamrzivaču.


Transdukcija bakterija

Genetska analiza termofilnih bakterija ograničena je na samo nekoliko vrsta: Bacillus stearothermophilus (Todlučiti se∼60 ° C), Thermus thermophilus (Todlučiti se∼70 ° C), Thermoanaerobacter vrsta (Todlučiti se∼60 ° C), i Thermotoga vrsta (Todlučiti se∼80 ° C). U svim slučajevima, dostupni genetski alati mnogo su manje sofisticirani od onih koji se koriste za proučavanje mezofila poput njih E coli. Za uvođenje DNA u bakterije koriste se tri metode: transdukcija, konjugacija i transformacija. Transdukcija je prijenos DNA posredstvom bakterijskog virusa (bakteriofaga) koji sadrži segment genomske DNA uklonjen iz prethodnog domaćina. Iako su poznati bakteriofagi koji inficiraju neke termofile, nije poznato da niti jedan nije sposoban prenijeti kromosomske gene na zaraženog domaćina. Konjugalan prijenos DNA između stanica uključuje kontakt stanica do stanice tijekom procesa prijenosa, a među nekima je primijećen Thermus sojeva u laboratoriju. Nije dokazano da to rade drugi termofili. Konačno, transformacija, proces u kojem stanice preuzimaju golu DNA, događa se u svim gore navedenim termofilima. Međutim, samo Thermus vrste se podvrgavaju "prirodnoj" transformaciji pri čemu stanice preuzimaju golu DNK tijekom rasta bez prethodne obrade tih stanica u laboratoriju. Ostali organizmi moraju biti prisiljeni preuzeti DNK prethodnom kemijskom obradom stanica ili ubacivanjem DNA u stanice pomoću električne sile (elektroporacija).


Opcije pristupa

Omogućite potpuni pristup časopisu 1 godinu

Sve cijene su NETO cijene.
PDV će biti dodat kasnije prilikom plaćanja.
Obračun poreza bit će dovršen tijekom plaćanja.

Nabavite vremenski ograničen ili potpuni pristup članku na ReadCube -u.

Sve cijene su NETO cijene.


KONTROLA REPLIKACIJE PLASMIDA

Iako se broj kopija plazmida može razlikovati kod različitih bakterija, unutar datog domaćina i pod stalnim uvjetima rasta, svaki određeni plazmid ima karakterističan broj kopija. To se postiže kontrolnim elementima kodiranim plazmidima koji reguliraju početak koraka replikacije. Kontrolni sustavi održavaju brzinu replikacije u ustaljenom stanju u prosjeku jedan replikacijski događaj po kopiji plazmida i staničnom ciklusu ispravljajući odstupanja od prosječnog broja kopija u pojedinim stanicama. Za definiranje i održavanje broja kopija, plazmidi koriste negativna regulatorna kola (251). Genetska analiza mehanizama kontrole replikacije prvo je izvedena za plazmid R1 izolacijom mutanata koji su pokazali povećan broj kopija (225). Karakterizacija ovih mutanata pokazala je da su odrednice kontrole broja kopija stanovale u samom plazmidu i da su negativni regulatori (inhibitori), djelujući u koraku inicijacije, uključeni u ovu kontrolu. Model kontrole replikacije moduliran negativnim efektorima prvi su predložili i kvantitativno opisali Pritchard i sur. (252). Kad plazmid kolonizira novog domaćina, koncentracija ovih negativnih regulatora trebala bi biti zanemariva. Čini se da je to poželjno za uspješnu uspostavu, jer bi neometana replikacija plazmida omogućila da se u kratkom vremenu postigne normalni broj kopije. Međutim, nakon što se postigne karakterističan broj kopija, održavanje prosječnog broja kopija u populaciji zahtijeva prilagodbe fluktuacija ove vrijednosti u pojedinim ćelijama. Kontrolni sustavi čine upravo to povećanjem ili smanjenjem brzine replikacije po kopiji plazmida i staničnom ciklusu. Pojedinačne kopije plazmida odabrane su za nasumičnu replikaciju iz grupe koja uključuje replicirane i nereplicirane kopije. Međutim, postoje mehanizmi koji suprotno odabiru novo replicirane molekule, npr. Hemimetilacija i supermotacija (3, 224).

Kontrola replikacije negativnim regulatorima i nasumični odabir plazmida za replikaciju imaju dodatnu posljedicu: dva plazmida s identičnim replikonima ne mogu stabilno koegzistirati unutar određene stanice u odsutnosti selektivnog tlaka. To dovodi do segregacije plazmida unutar populacije domaćina, fenomena poznatog kao nekompatibilnost plazmida. Inhibicija replikacije plazmida, povezana s povećanjem doze gena gena za kontrolu broja kopija, korištena je za identifikaciju ovih gena (pregledano u referencama 12, 51, 155, 222, 227 i 251).

Kontrola replikacije putem inhibitora zahtijevala bi da mogu "izmjeriti" koncentraciju kopija plazmida unutar stanice. To se može postići nestabilnim inhibitorom izraženim konstitutivno ili stabilnim inhibitorom sintetiziranim ubrzo nakon svakog inicijacijskog događaja (252). Bilo koja od ovih alternativa smanjila bi učestalost pokretanja nakon svakog inicijacijskog događaja te bi povećala ili smanjila brzinu započinjanja replikacije kada je prosječni broj kopija manji ili veći od potrebnog. Kad je učestalost inicijacije određena razinom inicijatorskog proteina, jedan mehanizam za kontroliranu replikaciju plazmida (za razliku od replikacije faga) bio bi inaktiviranje inicijatorskog proteina nakon svakog događaja replikacije (255, 319).

Mehanizmi koji kontroliraju replikaciju proučavani su u različitim sustavima (pregledano u referenci 293a), a otkriveno je nekoliko vrsta inhibitora: (i) antisense RNA (ColE1, R1 i pT181) (ii) i antisens RNA i protein (pMV158 i pIP501) i (iii) DNA mjesta za vezivanje proteina inicijatora (F, P1, RK2 i R6K). Shematski prikaz ovih regulatornih petlji prikazan je na sl. ​ Slika 9. 9. Kontrola replikacije proteinom (lambda-dv) dobro je dokumentirana i kvantificirana (222), ali nije opisana u prirodnim plazmidima i ovdje se ne raspravlja. Također se ne raspravlja o pomoćnim elementima koji također mogu modulirati replikaciju, ali koji nisu izravno uključeni u kontrolu učestalosti inicijacije.

Kontrola replikacije plazmidne DNA. Prikazani su primjeri kontrole pomoću Rep DNA mjesta vezanja (plazmid P1), antisens RNA (plazmidi R1 i pT181) i dvostrukim sustavom (plazmid pMV158). Transkripti su prikazani kao kontinuirane linije, sa strelicama koje pokazuju smjer sinteze. mRNA su prikazane debljim linijama od antisensnih RNA koje su označene kontratranskribiranim RNA (tanke linije s malim vrhovima strelica). Ostali simboli kako slijedi: čvrste elipse, pravokutnici podrijetla, promotori a.r.b.s., paratične okomite crte vezane za mjesto vezivanja ribosoma, interakcije mRNA-ctRNA plus, pozitivne interakcije minus i inhibitorne interakcije RNA-RNA ili protein-DNA.

Kontrola pomoću antisense RNA

Interakcije između RNA mogu modulirati dostupnost ili pRNA ili razinu Rep proteina koji ograničava brzinu, a koji su bitni za replikaciju. Antisense RNAs koje kontroliraju broj kopije plazmida komplementarne su regiji na 5 ′ kraju ciljnog transkripta (preprimer RNA za replikaciju ili rep mRNA) i nazivaju se �ntratranskribirane RNA ” (ctRNA) (227, 228, 309). U nekim slučajevima, inhibicija ctRNA postiže se u području uparivanja s ciljnom RNA. U drugim slučajevima, stvaranje dupleksa inhibitor-cilja dovodi do konformacijske promjene u ciljnom transkriptu, koja se čini neaktivnom. Primjeri ovih mehanizama kontrole opisani su u nastavku.

Kontrola obrade primarne RNA: plazmid ColE1.

ColE1 je bio prvi primjer gdje je utvrđeno da kontrolu replikacije vrši RNA (170, 296). Zapravo, analize kontrolnih krugova broja kopija ColE1 dovele su do identifikacije antisensnih RNA, otkrića u molekularnoj biologiji. trans-antisensne RNA koje djeluju dopuštaju utišavanje specifičnih gena uparivanjem s molekulama mRNA. Osnove kontrole replikacije ColE1 dobro su poznate (pregledano u referencama 47 i 82). Započinjanje replikacije započinje transkriptom od 550 nukleotida, RNA II, koji izrađuje RNAP, a posebno ga obrađuje RNaza H. Ova je obrada potrebna za prijelaz RNA u sintezu DNA, budući da stvara 3 ′-OH kraj na kojem Pol I započinje sintezu vodećeg lanca. Dostupnost ovog 3 ′-OH kraja ograničava brzinu iniciranja i modulirana je RNA od 108 nukleotida, antisens RNA I, koja je u potpunosti komplementarna s regijom na 5 ′ kraju pRNA II. Hibridno stvaranje između antisense i preprimer RNA mijenja ukupnu sekundarnu strukturu preprimera. Kao posljedica ove konformacijske promjene, 3 ′ kraj preprimera ne može se hibridizirati s DNA. To, pak, inhibira početak replikacije jer, u nedostatku hibridne DNA-RNA, preprimer ne obrađuje RNaza H. Genetske i biokemijske analize pokazale su da dolazi do interakcije antisensne RNA s komplementarnim sekvencama preprimera u početku u regijama izloženim kao jednolančane petlje na obje presavijene molekule RNA. Formiranje ovog početnog kontakta (ili kompleksa “kissing ”) je korak interakcije koji ograničava brzinu i dovodi do potpunog žarenja antisensne RNA I s predprimernom RNA II, u procesu koji počinje na 5 ′ kraj RNA I. RNA I se sintetizira iz konstitutivnog promotora (stoga je njegova razina proporcionalna broju kopije plazmida) i nestabilna je. Pomoćnu ulogu ima mali bazični protein, nazvan Rop ili Rom (276, 304). Rop stupa u interakciju s antisense RNA i s preprimerom, povećavajući učinkovitost stvaranja kompleksa “kissing ” i time smanjujući učinkovitost replikacije. Gen koji kodira ovaj protein nije odrednica nekompatibilnosti, ali može pojačati antisense RNA I-posredovanu inkompatibilnost.

Kontrola broja kopije plazmida R1.

Kontrola broja kopija u R1 provodi se na razini sinteze RepA, koja je modulirana proizvodima gena za kontrolu broja kopija, copB i pandur (pregledano u referencama 224 i 309). Proizvodi od copB i od pandur inhibirati repA ekspresiju na transkripcijskoj i posttranskripcijskoj razini. RepA ograničava brzinu replikacije, ponajprije djeluje u cis, i ne mogu se ponovno koristiti. Glavni kontrolni gen za broj kopija je pandur, dok copB igra pomoćnu ulogu. pandur je transkribiran iz konstitutivnog promotora, a njegov proizvod je nestabilna (poluvijek, 2 minute) RNA, CopA, koja je komplementarna vodećoj regiji (koja se naziva CopT) repA mRNA. Hibridna formacija između CopA i njenog cilja CopT također se postiže stvaranjem kompleksa “kissing ”. CopA-CopT hibrid inhibira sintezu RepA ometajući translaciju gena za kodiranje vodećeg peptida, slavina, koji je translacijski povezan repA (26). Učinkovitost inhibicije ovisi o afinitetu formiranja kompleksa poljupca (pregledano u referenci 226). Mutacije u pandur koji smanjuju učinkovitost stvaranja kompleksa povećavaju broj kopija, ali je replikacija još uvijek kontrolirana. Međutim, potpuna inaktivacija CopA dovodi do nekontrolirane (odbjegle) replikacije (pregledano u referencama 224 i 226). Dupleks CopA-CopT specifično se cijepa pomoću RNaze III, a čini se da je to cijepanje važan korak u kontroli sinteze RepA (25). Mutacijska analiza provedena na pandur pokazala je da je struktura glavne stabljike u CopA važna za njezinu funkciju. Prisutnost ispupčenja u području stabljike CopA potrebna je za brzo stvaranje dupleksa s CopT metom in vitro, kao i za učinkovitu inhibiciju in vivo (126). Osim toga, čini se da prisutnost ispupčenih nukleotida u stablu CopA rezultira strukturnom destabilizacijom koja igra zaštitnu ulogu protiv razgradnje RNazom III (125). Strukturne i funkcionalne studije o CopA omogućile su nam izvlačenje izravne povezanosti između doze gena (broj kopije plazmida) i razine CopA. To određuje konstantnu brzinu replikacije, neovisno o broju kopija plazmida ili stopi rasta domaćina. Iz ovih rezultata proizlaze dvije posljedice: (i) učestalost replikacije plazmida po kopiji je ϡ kada je broj kopija plazmida po ćeliji ispod prosjeka i ρ kada je broj kopija iznad prosjeka i (ii) broj kopija R1 raste sa smanjenjem stope rasta domaćina. Konstantna stopa replikacije po ćeliji, neovisno o broju kopija, polako će ispraviti odstupanja od prosječne učestalosti replikacije plazmida (jedna po kopiji plazmida po staničnom ciklusu), pa će stoga aktivno održavati konstantu prosječnog broja kopija (222). Drugi regulatorni element u R1, protein CopB, u normalnim okolnostima ima pomoćnu ulogu. Ovaj mali dimerni i bazični protein potiskuje transkripciju repA od promotora, P2, koji se nalazi nizvodno od copB (Sl. ​ (Sl.9). 9). U stacionarnim uvjetima, CopB je prisutan u zasićenim koncentracijama, potpuno potiskujući transkripciju iz P2 do bazalne razine. Pod tim uvjetima, repA mRNA se sintetizira uglavnom kao polikistronska copB-tap-repA RNK. Međutim, pod uvjetima u kojima broj kopija opada ili je nizak zbog ranih faza uspostavljanja plazmida, represija posredovana CopB nije učinkovita. U tim okolnostima, P2 promotor je smanjen i repA također se prepisuje kao a slavina-repA mRNA. To dovodi do prolaznog povećanja stope transkripcije za repA i, kao posljedica toga, do privremenog povećanja učestalosti replikacije plazmida. Dakle, derepresija P2 promotor bi imao važnu ulogu tijekom rane faze uspostave plazmida ili u situacijama u kojima broj kopija padne ispod razine divljeg tipa (224, 320). Međutim, računalna simulacija sugerirala je da bi pomoćna kontrolna petlja posredovana CopB-om trebala imati samo marginalni učinak, ne samo u stacionarnim uvjetima, već i tijekom uspostave plazmida (259).

Drugi slučajevi kontrole antisense RNA.

Postoje i drugi primjeri u kojima replikaciju kontrolira antisense RNA. Na primjer, u plazmidima grupa IncI α i IncI β, kontrola replikacije modulirana je kratkom ctRNA koja inhibira, kao u R1, sintezu Rep proteina koji ograničava brzinu, a koji je pak translacijski povezan sinteza vodećeg peptida. Inhibicija ctRNA vrši se na posttranskripcijskoj razini ometanjem sinteze vodećeg peptida. Osim toga, ctRNA sprječava stvaranje aktivatorske RNA pseudoknoze koja je potrebna za učinkovitu sintezu Rep proteina (pregledano u referenci 309).

Antisense RNA uključena je u utišavanje ori-γ plazmida R6K (243, 244). Replikacija iz ovog podrijetla zahtijeva sintezu aktivatorske RNA koja započinje unutar sedam iterona podrijetla i utišava je antisensna RNA. Čini se da protein inicijator replikacije, π, pogoduje interakcijama između ovih transkripata. Ovaj regulatorni sustav neovisan je o onom koji moduliraju iteroni. R6K nije jedini plazmid koji sadržava iteron koji se replicira u teti i u kojem antisens RNA igra ulogu u regulaciji replikacije. U plazmidu ColE2, ctRNA komplementarna lideru rep mRNA je uključena u kontrolu replikacije na posttranskripcijskoj razini generiranjem hibrida ctRNA-mRNA koja sekvestrira sekvence (e) bitne za sintezu Rep osim SD regije (287, 323).

Izravna inhibicija sinteze Rep: blokiranje rep prijevod.

Izravni način kontrole broja kopije plazmida pomoću ctRNA je inhibicija sinteze Rep blokiranjem pristupa ribosoma rep sekvence inicijacije translacije mRNA. Ova vrsta kontrole predložena je za neke plazmide koji se repliciraju mehanizmima pomaka niti (R1162) (154) i RC replikacije (pMV158) (67). U oba slučaja glavni inc determinanta se nalazi u regiji koja kodira ctRNA, a pretpostavljeni signali za inicijaciju rep prijevodi se postavljaju na predviđenu petlju rep mRNA. U slučaju R1162/RSF1010, učestalost inicijacije određena je razinom proteina koji veže RepC DNA (112, 153). Izražavanje od repC je reguliran na transkripcijskoj razini prvim genom repC operon (179) i na translacijskoj razini ctRNA koja je komplementarna signalima inicijacije translacije repC (154). Koji od ova dva regulatorna mehanizma ograničava brzinu sinteze RepC -a još nije dobro shvaćeno. U plazmidu koji se replicira s RC pMV158, predložena je ctRNA koja inhibira translaciju repB gen izravnom interakcijom s inicijacijom translacijskih sekvenci prisutnih u repB mRNA (67, 71, 72). Sekundarne strukture u ctRNA, osim one koja odgovara transkripcijskom terminatoru, nisu pronađene. Plazmidi kojima nedostaje regija DNA koja kodira ovaj terminator i komplementarna struktura na policajac mRNA su još uvijek osjetljive na ctRNA divljeg tipa isporučene u trans, sugerirajući da stvaranje kompleksa za ljubljenje može biti važno, ali nije bitno za stvaranje hibridne ctRNA-mRNA. Složenija regulatorna petlja u pMV158, koja uključuje zajedničko sudjelovanje ctRNA i represorskog Cop proteina, opisana je kasnije (vidi dolje). Analogne genetske strukture u regiji kontrole replikacije pronađene su u plazmidima obitelji pMV158 (69). Predložen je i dvokomponentni sustav (Cop protein i antisense RNA) koji kontrolira replikaciju plazmida pIP501 (35 vidi dolje). Za plazmid pUB110 čini se da sintezu inicijatorskog proteina RepU kontroliraju dvije antisensne ctRNA koje ometaju repU prijevod, budući da su navodne ctRNA komplementarne repU iniciranje translacijskih sekvenci (178), iako je također predložen mehanizam kontrole sličan onom pT181 (11, 249). No, budući da je sinteza RepU autoregulirana (207), možda će se morati razmotriti složenija razina kontrole.

Slabljenje transkripcije: paradigma pT181.

Mehanizam kontrole broja pojedinačnih kopija, koji uključuje ctRNA, dobro je uspostavljen za pT181 (119, 169, 227, 228). Trenutni model za kontrolu replikacije pT181 (reference 227 i 228 i reference u njemu) predlaže da je sinteza Rep inicijatora ograničena s dvije male ctRNA (koje imaju isti 5 ′ kraj, ali različita 3 ′ kraja), koje su komplementaran neprevedenom 5 ′ kraju rep mRNA (44, 119, 227, 229). Početna tvorba kompleksa poljupca između ctRNA i rep mRNA bi se odvijala kroz nukleotide izložene u komplementarnim petljama ukosnica koje se mogu formirati na obje vrste RNA. Formiranje hibrida ctRNA-mRNA dovelo bi do konformacijske promjene u području mRNA daleko od područja komplementarnosti. Nova konfiguracija mRNA bila bi takva da nova ukosnica završava na A (U)6 mogao se formirati slijed. Ova sekundarna struktura nalikuje na Rho neovisan terminator transkripcije i stoga može skraćivati rep mRNA (230). Stoga je transkripcijsko slabljenje (kao što je pronađeno u kontroli mnogih biosintetskih operona) (161), umjesto blokiranja inicijacije translacije, mehanizam za kontrolu broja kopije pT181. Slična kontrola, preuranjenim prekidom rep mRNA potaknuta neizravnim djelovanjem ctRNA, predložena je za plazmide iz obitelji pIP501/pAM 㬡 (33). Za pIP501 pokazalo se da su koraci koji se događaju prije stabilnog formiranja dupleksa ctRNA-mRNA dovoljni za slabljenje sinteze mRNA (34).

Kontrola i transkripcijskim represivom i antisens RNA

Kontrola zajedničkim djelovanjem transkripcijskog represora i antisense RNA okarakterizirana je za plazmide R1, pMV158, a u novije vrijeme i pIP501, iako su pronađene razlike u ulozi komponenti (35, 69, 71, 72, 224 ). Kao što je gore opisano, glavnu kontrolu u R1 vrši ctRNA CopA, dok CopB ima pomoćnu ulogu. Zapravo, brisanje copB dovodi samo do umjerenog povećanja broja kopija plazmida, a copB gen kloniran u kompatibilan replikon ne pokazuje nekompatibilnost prema R1.

U slučaju pMV158, transkripcijski represor, CopG, veže se i potiskuje transkripciju s jednog promotora za oba copG i repB geni (66). Mutacije ili brisanja u copG dovesti do povećanja broja kopije plazmida (7), i copG pokazuje samo slabu inkompatibilnost prema plazmidima s replikonom pMV158 kada se klonira, pod vlastitim signalima transkripcije i translacije, u vektor kompatibilan s velikim brojem kopija (67). Drugi element uključen u kontrolu broja kopija je mala kontratranskribirana RNA, RNA II, koja je komplementarna regiji policajac mRNA između gena copG i repB. rnaII je jak inc odrednica kada se isporučuje u trans pri visokim dozama gena. Mutacije koje ukidaju sintezu ctRNA također dovode do povećanja broja kopija, ali replikaciju još uvijek kontrolira CopG. Međutim, u ovom slučaju uočene su velike fluktuacije u broju kopija unutar pojedinih stanica (71). Nije pronađena značajna inkompatibilnost prema plazmidima s replikonom pMV158 kada je rnaII gen je kloniran u plazmid s malim brojem kopija (71). Elementi upravljačkog kruga (copG i/ili rnaII) klonirane u fiziološkoj dozi gena (tj. u broju kopija sličnom broju plazmida divljeg tipa). U ovom slučaju, jaka nekompatibilnost prema pMV158 pronađena je kada je cijeli krug uključen u trans dok je bilo koja komponenta klonirana zasebno pokazala slabu (ktrnaII) ili ne (copG) nekompatibilnost. Hipoteza izvedena iz ovih pokusa bila je da kontrolu replikacije pMV158 vrši cijeli regulatorni krug, a ne hijerarhija bilo koje komponente (71). Zbog genetske organizacije plazmida obitelji pMV158, čini se da u svima postoji sličan regulatorni krug (61).

Čini se da theta-replicirajući plazmid pIP501 dijeli značajke s R1 i pMV158 u njihovom kontrolnom krugu replikacije. Slično kao i R1, protein CopR pIP501 nije regulator vlastite sinteze, već potiskuje transkripciju iz esencijalnog nizvodnog rep promotor (31). Međutim, za razliku od R1, CopR inhibiran rep promotor nije u potpunosti potisnut, situacija nalikuje pMV158. Drugi element koji regulira broj kopije pIP501 je neobično dugovječna antisens RNA III (35). Stabilnost ove antisense RNA III predstavlja problem korekcije pada fluktuacija u broju kopija, budući da smanjenje broja kopija plazmida neće biti brzo praćeno istovremenim smanjenjem razine inhibitora. To bi dovelo do gubitka plazmida, što nije primijećeno (139a). Kako bi se riješio ovaj prividni paradoks, model sličan onom koji je predložen za pMV158 (71), u kojem bi smanjenje broja kopija pIP501 dovelo do deresije rep postavljen je promotor zbog smanjenja razine CopR (35).

Nedavno otkriće da protein RepU plazmida pUB110 regulira vlastitu sintezu, uvodeći tako dodatnu kontrolu za broj kopije plazmida (207), predstavljao bi još jedan primjer dvostruke kontrole (transkripcijska i translacijska regulacija). Mehanički, situacije za pUB110 i pMV158 mogu se razlikovati, što može ukazati na općenitiju kontrolu mehanizma replikacije u tim plazmidima koji se repliciraju pomoću RC načina.

Kontrola putem Iterona

Rep proteini plazmida koji sadrže replikaciju theta replikovanih iterona ili su autoregulirani ili su pod kontrolom transkripcije, a iteroni igraju ulogu u kontroli broja kopija plazmida (pregledano u referencama 51, 155 i 223). Uloga iterona prvo je opisana za plazmid F, na temelju zapažanja da oriS-ovisna replikacija bila je inhibirana kada su iteroni oriS klonirani su na kompatibilnom plazmidu (295). Slične situacije pronađene su i za druge plazmide koji sadrže iteron, poput P1 ili R1162/RSF1010. Utvrđeno je da je stupanj inhibicije u P1 proporcionalan broju kloniranih iterona (238), dok je u R1162 inhibicija ukinuta prekomjernom proizvodnjom Rep proteina koji ograničava brzinu (153). Ta su zapažanja dovela do formuliranja modela titracije (303). Model je pretpostavio da protein Rep ograničava brzinu iniciranja i da itenovi titriraju protein Rep, čime se ograničava učestalost iniciranja. Paradoks se razvio kada je utvrđeno da su proteini Rep većine plazmida koji sadrže iteron autoregulirani, što implicira da bi se titracija mogla nadvladati derepresijom (49). Nadalje, u drugim sustavima (R6K i RK2), količina proteina Rep unutar stanice očito je bila prevelika da bi ograničavala zasićenje vezivanja iterona (88, 190). To je dovelo do modifikacije početnog modela i do prijedloga da su različiti oblici Rep proteina uključeni u autoregulaciju i iniciranje, tako da titracija inicijatora ne uzrokuje derepresiju (302). Razvijen je računalni model koji se prilagođava ovoj hipotezi (321).

Drugo rješenje paradoksa titracije/autoregulacije pruženo je modelom da su molekule Rep vezane za P1 iterone još uvijek sposobne potisnuti repA promotor (49). Model se temeljio na sposobnosti Rep proteina da se istovremeno vežu na dva iterona (i u P1 i u R6K) (204). Ovo svojstvo Rep proteina također je poslužilo kao osnova za novi model negativne kontrole replikacije pod nazivom "model##x0201csteric smetnje"##x0201d (238) ili "#x0201chachuffinging" (155, 191). Prema ovom modelu, kada se Rep proteini vežu i zasićuju iterone podrijetla, dolazi do inicijacije ako je broj kopija plazmida nizak. S povećanjem broja kopija, molekule Rep vezane za iterone jednog podrijetla počinju komunicirati sa sličnim kompleksima nastalim na drugim podrijetlima. Posljedica toga je da se molekule plazmida spajaju kroz Rep-Rep interakcije, uzrokujući steričku smetnju u funkciji oba podrijetla (ȁs vezane lisice ”). Parovi plazmida se očito odvajaju tijekom kasnijeg staničnog rasta, a inicijacijski potencijal pojedinačnih molekula se obnavlja. Prema ovom modelu, koncentracija iterona, a ne razina izraza Rep, određuje brzinu replikacije. Pitanje uloge autoregulacije ostaje otvoreno i nije dan jednostavan odgovor. Kao što je gore spomenuto, u RK2 i R6K, značajno smanjenje razine Rep proteina nema utjecaja na brzinu replikacije plazmida. Međutim, u P1, dvostruko smanjenje razine Rep proteina ukida replikaciju, a četverostruko povećanje može dovesti do inhibicije replikacije (238). Slično, u R6K, dvostruko povećanje koncentracije inicijatora inhibira inicijaciju. Ovi podaci ukazuju na to da je autoregulacija važna, barem u nekim slučajevima, za održavanje optimalne koncentracije proteina inicijatora. Nedavno je predloženo da bi posebice plazmidi u kojima inicijatori ograničavaju, titracija inicijatora i uparivanje inicijatora (stavljanje lisica) mogli djelovati zajedno (51).

Neki od replikona koji sadrže iterone u podrijetlu također sadrže iterone izvan ove regije, koji izražavaju nekompatibilnost i dodatno smanjuju brzinu replikacije (237). U načelu, vjerojatnost uparivanja iterona posredovanog Repom uvelike se povećava u tim replikonima, jer se to može dogoditi u cis petljom DNA i u raznim kombinacijama u trans. Budući da bi kontrolni sustavi trebali mjeriti broj kopije, a budući da je intramolekularno uparivanje neovisno o ovom parametru, ovo uparivanje ne može ispraviti odstupanja od prosječnog broja kopija. Međutim, intramolekularno uparivanje može regulirati razinu na kojoj djeluje kontrola kopiranja (raspravljano u referenci 223). Drugačije je pitanje jesu li iteroni unutar i izvan početne regije jednako učinkoviti kao kontrolni elementi replikacije. Najnoviji podaci o plazmidima RK2 i P1 ukazuju na to da iteroni izvan ori are more effective as negative regulators of replication than are iterons present in the origin region, probably due to differences in the quantitative and structural features of the two sets (5, 273). It has been proposed that in RK2 these auxiliary iterons stimulate initial pairing between origins. The data for P1 indicate that the concentration of the auxiliary iterons rather than their relative arrangements is the deterministic factor for replication control (51).

The role played by Rep proteins of iteron-containing plasmids in the control of replication and in autoregulation implies that specific rep mutations affecting protein-protein or protein-DNA interactions could increase the plasmid copy number. In R6K, copy-up mutations affecting the plasmid initiator protein have been obtained (87). Most of the copy-up mutations affected a 32-amino-acid region placed between the LZ motif and the putative DNA-binding domain of the π protein, a region that seems to be involved in high-order oligomerization of the initiator protein (199). Mutations that affect the LZ motif of the Rep protein and lead to copy-up phenotypes in pSC101 have been described (133). Copy-up mutants of plasmid RK2 containing mutations encoding TrfA variants defective in binding to DNA have been isolated, indicating that copy number control is modulated by TrfA-DNA interactions (46). Copy-up mutations in the trfA gene have also been selected as intragenic suppressors of thermosensitive trfA mutacije. Some of these copy-up mutations were proposed to reduce a strong protein-protein inhibitory interaction induced, at the restrictive temperature, by the thermosensitive mutation (114).

Hemimethylation and Regulation of Plasmid Replication

The heptamer repeats containing the methylation sites in the origin of replication of plasmid P1 constitute the target of the host protein SeqA, involved in sequestering hemimethylated oriC into the bacterial membrane (37). Methylation seems to regulate replication at two different levels. First, hemimethylation would exert a negative regulation through ori-membrane interactions. The newly replicated, hemimethylated DNA is sequestered in the cell membrane, thus preventing reinitiation. The sequestration is relieved as the hemimethylated DNA undergoes new methylation (51). At a second level, methylation increases the initiation efficiency both in vivo and in vitro. This stimulation could be due to enhanced bending or unwinding of the DNA or to recycling of initiators (3, 51).

Synopsis

Plasmid DNA replication occurs coupled to the cell cycle of the bacterial host in such a way that a fixed concentration of plasmids is maintained in the bacterial population. This is achieved by mechanisms that monitor the initiation frequency and adjust this frequency to an average of one replication per plasmid copy and cell cycle. The control of plasmid replication is plasmid encoded and is performed by molecules (antisense RNA, proteins, or DNA sequences) that inhibit the initiation of replication in a dose-dependent way. This inhibition can limit the level of active Rep protein, the availability of the primer for the leading strand, or the number of active origins. Control of plasmid replication may be exerted (i) by a single inhibitor, (ii) by a main controller and an auxiliary element, or (iii) by the concerted action of two inhibitors (a protein and an antisense RNA or two sets of iterons). In general, any model on control of plasmid replication should explain how the average plasmid copy number is maintained in a growing population and how fluctuations on this average are corrected.


Biology of Serpins

Sangeeta R. Bhatia , . Cliff J. Luke , in Methods in Enzymology , 2011

2.2.2 Generation of transgenic lines for purification of serpin targets

Plasmid DNA microinjection is an essential tool for C. elegans research and is a straightforward technique for incorporating exogenous DNA into worms ( Kimble et al., 1982 Mello and Fire, 1995 Stinchcomb et al., 1985 ). Transgenic animals are created by microinjection of the plasmid DNA directly into the distal arm of the gonad ( Berkowitz et al, 2008.). The plasmid DNA encoding a given gene of interest is usually coinjected with another plasmid that serves as a transformation marker, providing either a physical ( Kramer et al., 1990 ) or visual ( Gu et al., 1998 ) phenotype by which to selectively identify transgenic animals. The plasmid DNA undergoes recombination to form extrachromosomal arrays that contain multiple copies of the coinjected DNAs in transgenic animals produced via microinjection ( Mello et al, 1991.). However, extrachromosomal arrays are not passed on to all progeny. Therefore, to maximize the amount of serpin that can be purified, it is advisable to integrate the array into the genome of the animal. For purification of serpin:protease complexes, it is necessary to create transgenic lines expressing the TrAP tag::serpin fusion protein with the knockout allele, for example, TrAP::SRP-6 in the srp-6(ok319) knockouts. This will then maximize the yield of serpin:protease complexes obtained by eliminating the native serpin that will not be purified by the TAP procedure. The methods for creating transgenic lines and integration of the extrachromosomal array are discussed in detail in Chapter 13 .


What is Recombinant DNA Technology

Recombinant DNA technology is a molecular biology technique used to produce recombinant DNA molecules that carry the desired characteristic to a particular organism. Molecular cloning is the laboratory technique used to produce a large copy number of recombinant DNA coupled with PCR. The process of molecular cloning consists of seven steps as described below.

  1. Choice of host organism and cloning vector – The host organism is mainly bacteria. The choice of cloning vector depends on the choice of the host organism, the size of the foreign DNA fragment, and the level of expression.
  2. Preparation of vector DNA – The cloning vector is digested with restriction enzymes to make compatible ends with the foreign DNA fragment.
  3. Preparation of DNA to be cloned – The desired DNA fragment to be cloned can be amplified by PCR and digested with the restriction enzymes to generate compatible ends with the cloning vector.
  4. Creation of recombinant DNA – The digested cloning vector and the PCR fragment are ligated by treating with DNA ligase.
  5. Introduction of recombinant DNA into the host organism – The recombined DNA molecules are transformed into bacteria to obtain a large number of copies.
  6. Selection of transformed organisms – a selectable marker such as antibiotic resistance can be used to select the transformed bacteria in a culture.
  7. Screening for clones with desired DNA – The blue-white screening system, PCR, restriction fragment analysis, nucleic acid hybridization, DNA sequencing, and antibody probes can be used to screen the clones with desired DNA fragment.

The steps of recombinant DNA technology is shown in figure 1.

Figure 1: Recombinant DNA Technology


Method #4: A quick way

Colony screening with Polymerase Chain Reaction (PCR) is the most rapid initial screen to determine the presence of the DNA insert. Colony PCR involves lysing the bacteria and amplifying a portion of the plasmid with either insert-specific or vector-specific primers. If you need to determine the orientation of your insert, it is recommended to combine both types of primers for your analysis.

If selecting colony screening by PCR, make sure that your insert is shorter than 3 kb. Some PCR reagents will allow you to add a portion of your individual colony directly to a PCR master mix, with the remaining portions being used to inoculate a culture plate or liquid media with appropriate antibiotic for downstream applications.


Move over CRISPR, the retrons are coming

3D-model of DNA. Credit: Michael Ströck/Wikimedia/ GNU Free Documentation License

While the CRISPR-Cas9 gene editing system has become the poster child for innovation in synthetic biology, it has some major limitations. CRISPR-Cas9 can be programmed to find and cut specific pieces of DNA, but editing the DNA to create desired mutations requires tricking the cell into using a new piece of DNA to repair the break. This bait-and-switch can be complicated to orchestrate, and can even be toxic to cells because Cas9 often cuts unintended, off-target sites as well.

Alternative gene editing techniques called recombineering instead perform this bait-and-switch by introducing an alternate piece of DNA while a cell is replicating its genome, efficiently creating genetic mutations without breaking DNA. These methods are simple enough that they can be used in many cells at once to create complex pools of mutations for researchers to study. Figuring out what the effects of those mutations are, however, requires that each mutant be isolated, sequenced, and characterized: a time-consuming and impractical task.

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School (HMS) have created a new gene editing tool called Retron Library Recombineering (RLR) that makes this task easier. RLR generates up to millions of mutations simultaneously, and "barcodes" mutant cells so that the entire pool can be screened at once, enabling massive amounts of data to be easily generated and analyzed. The achievement, which has been accomplished in bacterial cells, is described in a recent paper in PNAS.

"RLR enabled us to do something that's impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously," said co-first author Max Schubert, Ph.D., a postdoc in the lab of Wyss Core Faculty member George Church, Ph.D. "RLR is a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, which eliminates the toxicity often observed with CRISPR and improves researchers' ability to explore mutations at the genome level."

Retrons: from enigma to engineering tool

Retrons are segments of bacterial DNA that undergo reverse transcription to produce fragments of single-stranded DNA (ssDNA). Retrons' existence has been known for decades, but the function of the ssDNA they produce flummoxed scientists from the 1980s until June 2020, when a team finally figured out that retron ssDNA detects whether a virus has infected the cell, forming part of the bacterial immune system.

While retrons were originally seen as simply a mysterious quirk of bacteria, researchers have become more interested in them over the last few years because they, like CRISPR, could be used for precise and flexible gene editing in bacteria, yeast, and even human cells.

"For a long time, CRISPR was just considered a weird thing that bacteria did, and figuring out how to harness it for genome engineering changed the world. Retrons are another bacterial innovation that might also provide some important advances," said Schubert. His interest in retrons was piqued several years ago because of their ability to produce ssDNA in bacteria—an attractive feature for use in a gene editing process called oligonucleotide recombineering.

Recombination-based gene editing techniques require integrating ssDNA containing a desired mutation into an organism's DNA, which can be done in one of two ways. Double-stranded DNA can be physically cut (with CRISPR-Cas9, for example) to induce the cell to incorporate the mutant sequence into its genome during the repair process, or the mutant DNA strand and a single-stranded annealing protein (SSAP) can be introduced into a cell that is replicating so that the SSAP incorporates the mutant strand into the daughter cells' DNA.

"We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities," said co-first author Daniel Goodman, Ph.D., a former Graduate Research Fellow at the Wyss Institute who is now a Jane Coffin Childs Postdoctoral Fellow at UCSF.

Another attraction of retrons is that their sequences themselves can serve as "barcodes" that identify which individuals within a pool of bacteria have received each retron sequence, enabling dramatically faster, pooled screens of precisely-created mutant strains.

To see if they could actually use retrons to achieve efficient recombineering with retrons, Schubert and his colleagues first created circular plasmids of bacterial DNA that contained antibiotic resistance genes placed within retron sequences, as well as an SSAP gene to enable integration of the retron sequence into the bacterial genome. They inserted these retron plasmids into E. coli bacteria to see if the genes were successfully integrated into their genomes after 20 generations of cell replication. Initially, less than 0.1% of E. coli bearing the retron recombineering system incorporated the desired mutation.

To improve this disappointing initial performance, the team made several genetic tweaks to the bacteria. First, they inactivated the cells' natural mismatch repair machinery, which corrects DNA replication errors and could therefore be "fixing" the desired mutations before they were able to be passed on to the next generation. They also inactivated two bacterial genes that code for exonucleases—enzymes that destroy free-floating ssDNA. These changes dramatically increased the proportion of bacteria that incorporated the retron sequence, to more than 90% of the population.

Name tags for mutants

Now that they were confident that their retron ssDNA was incorporated into their bacteria's genomes, the team tested whether they could use the retrons as a genetic sequencing "shortcut," enabling many experiments to be performed in a mixture. Because each plasmid had its own unique retron sequence that can function as a "name tag", they reasoned that they should be able to sequence the much shorter retron rather than the whole bacterial genome to determine which mutation the cells had received.

First, the team tested whether RLR could detect known antibiotic resistance mutations in E coli. They found that it could—retron sequences containing these mutations were present in much greater proportions in their sequencing data compared with other mutations. The team also determined that RLR was sensitive and precise enough to measure small differences in resistance that result from very similar mutations. Crucially, gathering these data by sequencing barcodes from the entire pool of bacteria rather than isolating and sequencing individual mutants, dramatically speeds up the process.

Then, the researchers took RLR one step further to see if it could be used on randomly-fragmented DNA, and find out how many retrons they could use at once. They chopped up the genome of a strain of E. coli highly resistant to another antibiotic, and used those fragments to build a library of tens of millions of genetic sequences contained within retron sequences in plasmids. "The simplicity of RLR really shone in this experiment, because it allowed us to build a much bigger library than what we can currently use with CRISPR, in which we have to synthesize both a guide and a donor DNA sequence to induce each mutation," said Schubert.

This library was then introduced into the RLR-optimized E coli strain for analysis. Once again, the researchers found that retrons conferring antibiotic resistance could be easily identified by the fact that they were enriched relative to others when the pool of bacteria was sequenced.

"Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other," said senior author George Church, who leads the Wyss Institute's Synthetic Biology Focus Area and is also a Professor of Genetics at HMS. "This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research."

Another feature that distinguishes RLR from CRISPR is that the proportion of bacteria that successfully integrate a desired mutation into their genome increases over time as the bacteria replicate, whereas CRISPR's "one shot" method tends to either succeed or fail on the first try. RLR could potentially be combined with CRISPR to improve its editing performance, or could be used as an alternative in the many systems in which CRISPR is toxic.

More work remains to be done on RLR to improve and standardize editing rate, but excitement is growing about this new tool. RLR's simple, streamlined nature could enable the study of how multiple mutations interact with each other, and the generation of a large number of data points that could enable the use of machine learning to predict further mutational effects.


Understanding the New Technologies for RNA Isolation/Extraction

The quest for new viral disease therapies and test kits has placed greater emphasis on RNA isolation and extraction. New technologies have been introduced that offer greater
simplicity, cost savings, and product availability over commercial kits, while achieving equivalent RNA quality and quantity. These technologies use single devices in addition to the laboratory’s leftover or preferred reagent kits.

One such technology, the nucleic acid extraction filter plate, can deliver high-quality isolation and purification of RNA and genomic DNA from mammalian cells at high throughput without sacrificing results. In cases where nucleic acid preparations require low to medium throughput, users frequently must employ kits containing expensive centrifugal devices that generate leftover reagent waste. Another new technology, the nucleic acid extraction spin column, can enable one device to perform these experiments with the laboratory’s own reagents.

High-throughput RNA purification with a nucleic acid extraction filter plate

The transcription of genes into RNA is an important step in the synthesis of functional gene products, which can be functional RNA species themselves or protein products formed after translation of messenger RNAs.

In many high-throughput gene expression analysis studies, cultured mammalian cells are exposed to different growth conditions. Reverse-transcription quantitative PCR (RT-qPCR) is used to measure the influence of the various conditions on the expression of genes of interest. Viral RNA isolation and extraction are essential prior to RT-qPCR in the detection of a virus in a sample.

This workflow is commonly performed in the development of vaccines and test kits for viral diseases. The studies are greatly aided by the availability of multiwell nucleic acid extraction filter plates with silica-based media that permit high-throughput total RNA isolation in a robust fashion.

Our study evaluated the performance of extraction filter plates versus commercially available RNA isolation kits. The filter plate contains a silica-based quartz glass fiber media for the isolation of total RNA from cultured mouse bEnd.3 endothelioma cells.

Robustness of the nucleic acid extraction filter plate as an RNA isolation medium was shown through the use of a protocol that relies on standard reagents readily obtained and prepared in-house by the researcher in an economically favorable manner. The latter protocolwas also implemented in an industrial process to isolate RNA from
mouse embryonic fibroblasts at a range of cell concentrations, and to
subject this RNA to one-step RT-qPCR assays for three genes.

Filter plate results

RNA isolation performance from cultured mouse bEnd.3 endothelioma cells was evaluated over a range of cell amounts employing two isolation protocols: the first with commercial reagents and the second with standard reagents prepared in-house.

Results of the RNA concentration measurements using the Quant-iT RiboGreen RNA assay are shown in Slika 1. The results indicate that regardless of whether commercial reagents or standard reagents were used with the nucleic acid extraction filter plate, RNA yields were similar to those obtained with the commercial kit.

Figure 1. RNA concentrations of samples isolated from 3.1–400× 10 3 cells were determined via Quant-iT RiboGreen RNA assay. Samples were isolated with a nucleic acid extraction filter plate using either a commercial reagent protocol (blue squares) or standard reagent protocol (blue triangles) or with a commercially available bundled plate/reagent kit (red squares).

RNA samples isolated with the nucleic acid extraction filter plate were separated on a DNA 5K/RNA/CZE LabChip in the LabChip GX II Touch HT instrument to determine sample integrity. The instrument’s GX Touch software further assessed RNA integrity by performing a smear analysis of the electropherogram traces. An internal algorithm was used to calculate the RNA Integrity Number (RIN). The electropherograms showed two distinct RNA peaks corresponding to 18S and 28S rRNA with little to no evidence of a smear to indicate RNA degradation (Figure 2A). For samples derived from 100–400 × 10 3 cells, RIN numbers varied from 8.0 to 9.6 (Figure 2B), indicating that the RNA was of high quality.

Figure 2. LabChip analysis demonstrating quality of bEnd.3 RNA isolated with the nucleic acid extraction filter plate. (A) LabChip electropherograms of RNA from 100–400 × 103 bEnd.3 cells isolated with either the commercial reagent protocol or the standard reagent protocol. (B) RNA integrity number.

RNA integrity alone does not guarantee successful amplification in an RT-qPCR application as copurified inhibitors can impede reverse transcription and/or PCR efficiency. Mouse bEnd.3 cells are characterized by the expression of VCAM-1 (vascular cell adhesion molecule 1, which is encoded by the Vcam-1 gene). The average Ct values showed a
linear inverse relationship with increasing bEnd.3 cell numbers. In samples
isolated with either protocol or plate, both the abundant GADPH (housekeeping protein glyceraldehyde-3-phosphate dehydrogenase, which is encoded by Gapdh) and the less abundant VCAM-1 message were detected. The slopes of the curves through points of RNA samples isolated from increasing numbers of cells were equal, indicating that no
inhibitory components copurified.

Figure 3. Expression of GAPDH and VCAM-1 in mouse bEnd.3 cells as determined via RT-qPCR. One-step RT-qPCR was carried out to detect messages encoded by the genes Gapdh and Vcam-1. The mouse bEnd.3 cell RNA samples were derived from 3.1–400 × 10 3 cells and isolated with nucleic acid extraction filter plates, using either a commercial reagent protocol (blue squares) or standard reagent protocol (blue triangles), or with a commercially available bundled plate/reagent kit (red squares).

Nucleic acids prepared with the extraction filter plate technology were of high quality and suitable for common downstream activities such as RT-qPCR, restriction digests, and sequencing (Slika 3).

Low- to medium-throughput RNA purification with a nucleic acid extraction spin column

The nucleic acid extraction spin column purifies RNA and genomic and plasmid DNA from bacteria, yeast, mammalian cultured cells, and plants. It is outfitted with an innovative duallayer, silica-based, quartz glass fiber matrix that enables its use with the buffers available in most currently available commercial kits.

Solid-phase extraction such as the spin-column-based method allows the nucleic acid to bind to the solid-phase matrix, 1,2 limiting the problems associated with liquid-liquid
extraction. 3 Solid-phase extraction facilitates the nucleic acid extraction process, making it fast, efficient, and reproducible compared with conventional methods. 2,3 The extraction device recovers RNA fragments ranging in size from 50 bp to 10,000 bp.

Spin column results

Reagents from commercially available kits (CK1) were used, and RNA from Escherichia coli bacterial cultures, mammalian cultured cells (CHO), and plant leaves (basil) were extracted and purified with the spin column. Kao što je prikazano u Lik 4, the nucleic acid yields recovered with the extraction spin column were comparable to those obtained with the spin devices from commercially available kits.

The data shows that the nucleic acid extraction spin column effectively extracts and purifies RNA and genomic DNA from common starting materials (stol 1).

The spin column has been thoroughly tested to assess performance with RNA extraction and purification from various starting materials. It proves that RNA (and genomic DNA) from bacteria, mammalian cells, and plants can be purified with this single device.

Moreover, the purified nucleic acids were processed in the most common molecular biology applications. The results demonstrated that nucleic acids purified with the extraction spin device meet the yield and purity requirements of the downstream applications.

Zaključci

The nucleic acid extraction filter plate and nucleic acid extraction spin column offer complementary options for high- and low-throughput nucleic acid purification. They are standalone, low-cost alternatives to purchasing full commercial kits. The technologies enable laboratories to perform RNA extraction and isolation with their own reagents and receive the RNA quantity and quality associated with commercial kits. These options are highly attractive at a time when viral research and development has placed a
premium on material availability.

Lori Euler ([email protected]) is the product manager for the molecular portfolio at Pall.

1. Ali N, Rampazzo RCP, Costa ADT, Krieger MA. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. Biomed. Res. Int. 2017 2017: 9306564. DOI:10.1155/2017/9306564.

2. Zwir-Ferenc A, Biziuk M. Solid phase extraction technique: Trends, opportunities and applications. Pol. J. Env. Klinac. 2016 15(5): 677–690.

3. Tan SC, Yiap BC. DNA, RNA, and protein extraction:The past and the present. J. Biomed. Biotehnol. 2009 2009: 574398. DOI: 10.1155/2009/574398.


Genetic engineering in animals

Some of the animals most extensively used as model systems for DNA manipulation are Caenorhabditis elegans (a nematode), Drosophila, and mice. Versions of many of the techniques considered so far can also be applied in these animal systems.

Transgenic animals.  

There are several ways of producing transgenic animals. An example is the production of transgenic Drosophila by the injection of plasmid vectors containing P elements into the fly egg (described in detail in Chapter 20). Transgenic Drosophila provide us with another illustration of the use of the bacterial lacZ gene as a reporter in the study of gene regulation during development. The lacZ gene is fused to the promoter region of a Drosophila heat-shock gene, which is normally activated by high temperatures. This construct is then used to generate transgenic flies. Subsequent to heat shock, the flies are killed and bathed in X-Gal. The resulting pattern of blue tissues provides information on the major sites of action of the heat-shock gene (Figure 13-19). Another approach to the production of transgenic mammals is by injecting special plasmid vectors into a fertilized egg (considered later in this chapter).

Figure 13-19

Transgenic Drosophila expressing a bacterial β-galactosidase gene. Drosophila was transformed with a construct consisting of the E. coli lacZ gene driven by a Drosophila heat-shock promoter. The resulting flies were heat shocked, killed immediately, (more. )

In both these cases, because it is the egg that is initially made transgenic, the extra DNA can find its way into germ-line cells, is then passed on to the progeny derived from these cells, and behaves from then on rather like a regular nuclear gene. Like plants, animals are being manipulated not only to improve the qualities of the animal itself, but also to act as convenient producers of foreign proteins. For example, mammalian milk is easily obtained, so it is a convenient medium for the collection of proteins that are otherwise more difficult to obtain without sacrificing the animal (Figure 13-20).

Figure 13-20

Production of a pharmaceutically important protein in the milk of transgenic sheep. The gene of interest encodes a protein that is of therapeutic importance, such as tissue plasminogen activator used to dissolve blood clots in humans. The gene is placed (more. )

Gene disruptions and gene replacement in mice.  

Mice are the most important models for mammals generally. Furthermore, much of the general technology developed in mice can be applied to humans. Two key techniques are the abilities to disrupt a gene (perhaps for a reverse genetics study) and to replace one allele with another. We shall consider examples of these techniques.

As mentioned earlier, gene disruptions are sometimes called knockouts. An organism carrying the gene knockout can then be examined for altered phenotypes. Knockout mice are invaluable models for the study of mutants similar to those found in humans. For instance, knockout mice that lack vital DNA repair enzymes (see Chapter 7) have been created to study whether these enzymes control cancer rates.

Figures 13-21 and 13-22 illustrate, step-by-step, the generation of a knockout mouse. First, a cloned, disrupted gene is used to produce embryonic stem (ES) cells containing a gene knockout (Figure 13-21a). Although recombination of the defective part of the gene into nonhomologous (ectopic) sites is much more frequent than its recombination into homologous sites (Figure 13-21c), selections for site-specific recombinants and against ectopic recombinants can be used, as shown in Figure 13-21d). Second, the ES cells that contain one copy of the disrupted gene of interest are injected into an early embryo (Figure 13-22). The resulting progeny are chimeric, having tissue derived from either recipient or transplanted ES lines. Chimeric mice are then mated to produce homozygous mice with the knockout in each copy of the gene (Figure 13-22).

Figure 13-21

The production of cells that contain a mutation in one specific gene, known as a targeted mutation or a gene knockout. (a) Copies of a cloned gene are altered in vitro to produce the targeting vector. The gene shown here has been inactivated by insertion (more. )

Figure 13-22

Producing a knockout mouse carrying the targeted mutation. (a) Embryonic stem (ES) cells (green at far left) are isolated from an agouti mouse strain and altered to carry a targeted mutation in one chromosome. The ES cells are then inserted into young (more. )

PORUKA

Fungal, plant, and animal genes can be cloned and manipulated in bacteria and reintroduced into the eukaryote cell, where they generally integrate into chromosomal DNA.

The technology for mammalian gene knockouts is similar to that for gene replacement. In gene therapy, a mutant allele is replaced by a wild type, the gene replacement providing a cure for the mutant condition.

U dogovoru s izdavačem, ovoj je knjizi dostupna značajka pretraživanja, ali je nije moguće pregledavati.