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Stabilan i jak promoter?

Stabilan i jak promoter?


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Trebam promotor sisara koji će održati stabilnu ekspresiju kroz diferencijaciju.

Prvobitno sam planirao da zaposlim UbC za ovaj specifičan projekat, međutim nove informacije iz drugog eksperimenta su izašle na videlo i sada mi treba promotor koji ima i ekstremnu stabilnost i veliku snagu (kao što znate, UbC je poznat po veoma niskoj ekspresiji stope).

Ima li neko od vas prijedloge? Možda bih trebao potražiti neku vrstu hibridnog promotera…

Hvala ti,

CDB


Čini se da postoji dosta sredstava za pomoć OP -u.

Addgene ima stranicu na temu sa popisom i opisom uobičajenih promotera koji se mogu koristiti u raznim organizmima: Plasmids 101: The Promoter Region

U eksperimentalnom udžbeniku biofizike spominju se i neki korisni promotori ili ovaj pregled.

Iznenađujuće, došlo je do neke kvantitativne analize jačine promotora: Sistematsko poređenje konstitutivnih promotora i promotora induciranog doksiciklinom, kao i napor ka jačem sintetičkom (verzija CMV-a) sintetičkom dizajnu jakih promotora

Moj zaključak je da bi virusni ili CAG (zasnovani na pilećem aktinu) promotori mogli biti korisni kandidati. Ali ne može se očekivati ​​sveprisutno i ravnomjerno izražavanje u različitim tipovima tkiva/ćelija bez finog podešavanja ekspresione kasete.


Stabilan i snažan promoter? - Biologija

Svi članci koje objavljuje MDPI odmah su dostupni širom svijeta pod licencom otvorenog pristupa. Nije potrebna posebna dozvola za ponovnu upotrebu cijelog ili dijela članka koji je objavio MDPI, uključujući slike i tabele. Za članke objavljene pod licencom Creative Common CC BY s otvorenim pristupom, bilo koji dio članka može se ponovno koristiti bez dopuštenja pod uvjetom da je izvorni članak jasno citiran.

Radovi predstavljaju najnaprednija istraživanja sa značajnim potencijalom za veliki uticaj na terenu. Radovi se dostavljaju na individualni poziv ili preporuku naučnih urednika i prolaze recenziju prije objavljivanja.

Značajni rad može biti ili originalni istraživački članak, značajna nova istraživačka studija koja često uključuje nekoliko tehnika ili pristupa, ili opsežan pregledni rad sa sažetim i preciznim ažuriranjima najnovijeg napretka u ovoj oblasti koji sistematski preispituje najuzbudljivija dostignuća nauke književnost. Ova vrsta papira daje pregled budućih pravaca istraživanja ili mogućih primjena.

Članci po izboru urednika temelje se na preporukama naučnih urednika MDPI časopisa iz cijelog svijeta. Urednici odabiru mali broj članaka nedavno objavljenih u časopisu za koje vjeruju da će biti posebno zanimljivi autorima ili važni u ovoj oblasti. Cilj je pružiti snimak nekih od najuzbudljivijih radova objavljenih u različitim istraživačkim područjima časopisa.


Grafički sažetak

Jesenji vojni crv, Spodoptera frugiperda, porijeklom iz Amerike, postaje svjetski štetočina koja nanosi štetu višestrukim usjevima (Jing et al., 2019.). S. frugiperda ćelijska linija, Sf9 razvijena je iz jajnika, a Sf21 odabran iz Sf9 naširoko je korišten za proizvodnju rekombinantnih proteina (Davis i sur., 1993. McCall i sur., 2005.). Prolazna transfekcija plazmidnih konstrukata u ove ćelije (Chang et al., 2018 Shen et al., 2014), selekcija stabilnih ćelija iz transficiranih ćelija (Fernandes et al., 2012 Kempf et al., 2002) i vektorski sistem ekspresije bakulovirusa (BEVS) (Smith et al., 1983) obično se koriste za proizvodnju rekombinantnih proteina u tim stanicama. Rani promotori virusnih gena, OpIE1 (Theilmann i Stewart, 1991.), OpIE2 (Theilmann i Stewart, 1992.) i hr5/ie1 (Jarvis et al., 1996) su najčešće korišteni promotori u ekspresiji na bazi plazmida S. frugiperda ćelije. Međutim, niži nivoi ekspresije stranih proteina predstavljaju ograničenje u korištenju ovih virusnih promotora (Chang et al., 2018 Zhao i Eggleston, 1999). Pokazalo se da endogeni promotori podržavaju visok nivo proizvodnje proteina u Drosophila melanogaster S2 ćelije (Angelichio et al., 1991). Dakle, visoko aktivni endogeni promotori iz S. frugiperda može povećati ekspresiju rekombinantnih proteina u S. frugiperda ćelijske linije. Visoko aktivni promotori kasnih virusnih gena, p10 i poliedar podržavaju visok nivo ekspresije proteina upotrebom bakulovirusa u S. frugiperda ćelija, stoga se ovaj sistem široko koristi za ekspresiju rekombinantnih proteina (Hill-Perkins i Possee, 1990). Međutim, do ekspresije rekombinantnih proteina dolazi u kasnoj fazi infekcije bakulovirusom kada je mehanizam sinteze proteina u stanicama domaćinima već oslabljen (Schultz i Friesen, 2009), što rezultira neefikasnom obradom i post-translacijskom modifikacijom rekombinantnih proteina ( Jarvis i dr., 1993. Jarvis i Summers, 1989. Schultz i Friesen, 2009.). Visoko aktivni endogeni promotori mogu olakšati ekspresiju rekombinantnih proteina u ranim fazama infekcije bakulovirusom, što rezultira poboljšanjima u obradi i post-translacijskim modifikacijama rekombinantnih proteina.

Promotori izvedeni od insekata domaćina uspješno su korišteni u poticanju ekspresije transgena kod modela insekata, uključujući Tribolium castaneum (Eckermann et al., 2018 Lorenzen et al., 2002 Rylee et al., 2018), Aedes aegypti (Anderson i sur., 2010. De Valdez i sur., 2011. Li i sur., 2011.), i Bombyx mori (Sakai et al., 2016 Tamura et al., 2000 Xu et al., 2019), što je pomoglo u osnovnim i primijenjenim istraživanjima ovih insekata. Nekoliko S. frugiperda Promotori su identifikovani transkriptomom i analizom genoma ćelija Sf21, međutim, nijedan od njih nije nadmašio rani virusni promotor OpIE2 u ćelijama Sf21 (Bleckmann et al., 2015). Prethodno smo identificirali nekoliko promotora induciranih toplinom koji su aktivni u ćelijama Sf9, a dva od njih su bila robusnija od OpIE2 promotor, ali manje aktivan od hr5/tj.1 promoter (Chen et al., 2020). Identificirati S. frugiperda promotori koji su aktivniji od komercijalno korištenih virusnih promotora, analizirali smo skup visoko izraženih gena iz transkriptoma dvaju S. frugiperda ćelijske linije i tri tkiva. Performanse potencijalnih promotera visoko izraženih gena procijenjene su u ćelijama Sf9 i Sf17. Promotori koji su pokazali veću aktivnost od komercijalno korištenog promotora (hr5/ie1) su dalje evaluirani prolaznom i stabilnom ekspresijom transgena u Sf9 ćelijama i FAW koristeći sisteme ekspresije baziranih na plazmidu i bakulovirusne ekspresijske sisteme, respektivno. Ove studije identificirale su dvije visoko aktivne S. frugiperda promoteri koji bi bili korisni za ekspresiju proteina, uređivanje genoma i proizvodnju transgenih insekata u FAW i drugim lepidopteranskim insektima.


Rezultati i rasprava

Skrining endogenih jakih promotora iz P. mendocina NK-01 putem RNA-seq analize i predviđanja promotora

Za RNA-seq analizu, nivo transkripcije gena je u pozitivnoj korelaciji sa RPKM vrijednošću 26. Kroz RNA-seq analizu P. mendocina NK-01, nivoi transkripcije svih gena rangirani su od visokog do niskog na osnovu njihovih vrijednosti RPKM. Pretpostavljeno je da je prvih 30 gena rangiranih prema RPKM vrijednostima visoko aktivno na transkripcijskom nivou (Tabela S1). Tako su uzvodna područja 30 gena s visokim vrijednostima RPKM odabrana kao ciljevi otkrivanja za predviđanje promotora. Daljnjim skriningom koristeći softver za predviđanje promotora na mreži, 10 od 30 sekvenci kandidata identificirano je kao pretpostavljena sekvenca promotora (slika S1) i odabrano za kasnije kloniranje i karakterizaciju (Tablica 1).

Kloniranje jakih promotera iz P. mendocina NK-01

Promotorske regije 10 visoko eksprimiranih gena PCR su amplifikovane iz genomske DNK P. mendocina NK-01. Da bi se dobila intaktna promotorska sekvenca svakog od 10 visoko eksprimiranih gena, u ovom je radu cijela međugenička regija između visoko eksprimiranog gena i njegovog gena uzvodno odabrana kao ciljna regija koju treba klonirati PCR -om, osim prirodnog vezivanja ribosoma sajt (RBS). Rezultati sekvenciranja DNK pokazali su da se klonirani fragmenti DNK podudaraju s odabranim međugenim regijama na razini nukleotida (podaci nisu prikazani).

Karakterizacija kloniranih promotora putem qPCR -a

Da bi se procijenila snaga kloniranih promotora, sekvence promotora su spojene na 5′-kraj amplificiranog gfp gen, a zatim umetnut u vektor za kloniranje širokog raspona domaćina pBBR1MCS-2 koji je sposoban da se replicira u različitim gram-negativnim bakterijama 27 koristeći homolognu rekombinaciju (slika 1). qPCR je korišten za analizu nivoa transkripcije gfp pod različitim promotorima u različitim fazama rasta, tj. ranoj log-fazi (6 h), post log-fazi (12 h) i stacionarnoj fazi (15 h) (slika S2). Među 10 testiranih promotora, nivoi transkripcije pet promotora P4, P6, P9, P16 i P25 bili su mnogo veći od nivoa transkripcije lac promoter u različitim fazama rasta. U odnosu na lac promotor, najjači promotor P4 je pokazao 36 puta povećanje transkripcione aktivnosti u stacionarnoj fazi (slika 2). Prilikom detekcije s većinom kloniranih promotora, nivoi transkripcije reporterskog gena gfp značajno varirala u različitim fazama rasta. Pet jakih promotora P4, P6, P9, P16 i P25 imalo je više nivoe transkripcije u post log-fazi nego u stacionarnoj fazi ili ranoj log-fazi (slika S3). Nasuprot tome, relativno male razlike u nivoima transkripcije otkrivene su sa pet jakih promotora između stacionarne faze i rane log-faze (slika S3). Promotor P16 imao je relativno stabilnu transkripcionu aktivnost tokom perioda rasta (slika S3). U prethodnim studijama različiti tipovi promotora, uključujući jake promotore, promotore ovisne o fazi rasta i konstitutivne promotore, dobro su okarakterizirani 10,13. Zbog dobre kompatibilnosti sistema sa ćelijom domaćinom, izbor endogenih promotora može biti praktičniji i svrsishodniji za njihovu primjenu u sintetičkoj biologiji i metaboličkom inženjeringu samog domaćina.

Rekombinantni plazmidi za karakterizaciju promotora sa gfp kao reporterski gen. (a) Rekombinantni plazmid sa a lac promoter kao kontrola. (b) Rekombinantni plazmidi za karakterizaciju snage 10 odabranih endogenih promotora.

Karakterizacija odabranih promotera i lac promoter putem qPCR analize. Transkripcija gfp gen pod različitim promotorima u P. mendocina NKU je kvantificiran u različitim fazama rasta. Kao interna referenca korišten je gen 16S rDNA. Relativna vrijednost transkripcije od gfp gen ispod lac promotor je postavljen na 1. Podaci predstavljaju srednje vrijednosti ± standardne devijacije trostrukih mjerenja iz tri nezavisna eksperimenta. Studentski t-test je obavljen između lac promoter i izabrani promoteri. * i ** označavaju P & lt 0,05 i P & lt 0,01, respektivno.

Promotori P17, P18 i P29 imali su visoke vrijednosti RPKM u RNA-seq analizi, ali su promotori postavljeni na plazmid pokazali niže nivoe transkripcije od lac promotora u bilo kojoj fazi rasta (slika 2). Odabrani endogeni promotori dobro funkcionišu u genomu, što se može pripisati pomoći obližnjih regulatornih sekvenci. Kada su promoteri odvojeno klonirani, nisu dobro funkcionisali.

Budući da se očekivalo da će se skrinirani endogeni promotori koristiti za poboljšanje proizvodnje PHA, RNA-seq podaci su dobiveni sa P. mendocina uzgaja se u PHA podlozi za fermentaciju. Za karakterizaciju kloniranih promotora testom reporterskog gena, za ovu studiju je odabran i uobičajen LB medijum za različite testove reporterskih gena 11,13. Dobro okarakterisani promotori u LB medijumu takođe mogu imati potencijal da se primene za sintezu drugih proizvoda u P. mendocina. Međutim, nivoi transkripcije 10 promotera kandidata dobijeni analizom RNA-seq ne moraju uvijek biti u skladu s nivoima transkripcije mjerenim qPCR-om zbog različitih uslova kulture. Na primjer, promotori P17, P18 i P29 su imali visoke vrijednosti RPKM u RNA-seq analizi, ali su pokazali niže nivoe transkripcije u testu reporterskog gena od lac promoter u bilo kojoj fazi rasta. U budućnosti bi se trebalo uzeti u obzir više RNA-seq podataka zasnovanih na različitim uslovima kulture za odabir kandidata za promotere. Zatim, kroz karakterizaciju navodnih promotora testom reporterskog gena u LB mediju, pregledani jaki endogeni promotori mogu imati široku primjenu za različite proizvode u P. mendocina.

Karakterizacija kloniranih promotora mjerenjem fluorescencije pomoću GFP -a

Kako bi se dalje odredili nivoi ekspresije odabranih endogenih promotora, mjereni su relativni intenziteti fluorescencije u tri različite faze rasta. Kao što je prikazano na slici 3, pet jakih promotora P4, P6, P9, P16 i P25 koje karakteriše qPCR imali su i veći relativni intenzitet fluorescencije od lac promoter u bilo kojoj fazi rasta. P4 je imao najjači relativni intenzitet fluorescencije među 10 odabranih promotora, što je pokazalo gotovo 32-struko povećanje u odnosu na lac promotor u stacionarnoj fazi. Za svakog od gore navedenih pet snažnih promotora, uočena je značajna razlika u intenzitetu fluorescencije GFP -a u različitim fazama rasta, što je u skladu s prethodnim rezultatima o nestabilnim nivoima transkripcije gfp mjereno qPCR (slika S3). Svi rezultati sugeriraju da razine ekspresije pet jakih promotora možda nisu konstantne tijekom cijelog ciklusa rasta (slika 3). Redovi snage promotora odraženi u realnom vremenu qPCR i GFP reporter bili su identični dobivenim rezultatima pomoću RNA-seq (RPKM vrijednost), što je pokazalo da su rezultati analize sekvenciranja transkriptoma i eksperimenata funkcionalne validacije bili vrlo konzistentni.

Karakterizacija odabranih promotora i lac promotor putem merenja intenziteta fluorescencije GFP. Izražavanje gfp gen pod različitim promotorima u P. mendocina NKU je kvantificiran u različitim fazama rasta. Pozadinski izraz je oduzet, a relativni intenzitet fluorescencije izračunat je normalizacijom prema OD600 celih ćelija. Podaci predstavljaju srednje vrijednosti ± standardne devijacije trostrukih mjerenja iz tri nezavisna eksperimenta. Studentski t-test je obavljen između lac promoter i odabrani promoteri. * i ** označavaju P & lt 0,05 i P < 0,01, respektivno.

Zanimljivo je da je relativni nivo transkripcije P25 bio veći od onog u P6, P9 i P16 (slika 2), ali je relativni intenzitet fluorescencije P25 bio manji od onog u P6, P9 i P16 (slika 3). Relativni intenziteti fluorescencije preostalih promotora P17, P18, P20, P23 i P29 bili su niži od intenziteta lac promoter u bilo kojoj fazi rasta, iako su P20 i P23 pokazali viši nivo transkripcije od lac promoter. Opažanja sugeriraju da visok nivo transkripcije gena ne mora nužno dovesti do visoke razine sinteze ovog proteina kodiranog genom. Da bi se održala konzistentnost efikasnosti inicijacije translacije, u ovoj studiji, isti RBS je uveden u uzvodno od reporterskog gena gfp. Među vektorima reporterskih gena, udaljenost između predviđenih promotorskih sekvenci i RBS -a bila je različita jedna od druge, što može utjecati na efikasnost translacije mRNA, što može dovesti do neslaganja između nivoa transkripcije i intenziteta fluorescencije. Na primjer, udaljenost između predviđenih promotorskih sekvenci i RBS-a za P25 bila je veća od one za P6, P9 i P16. Ovo može biti razlog zašto je P25 imao viši nivo transkripcije, ali niži intenzitet fluorescencije od onih kod P6, P9 i P16. Osim toga, razlike u spejser sekvencama između promotora i RBS -a mogu biti drugi razlog za različite trendove između nivoa transkripcije i intenziteta fluorescencije.

Kad se promatra konfokalnom mikroskopijom, stanice se eksprimiraju gfp pod kontrolom P4, P6, P9, P16 i P25 proizvodile su više svetlo zelene fluorescencije od kontrolnih ćelija sa gfp izraz pod kontrolom lac. Ćelije su proizvodile slabu zelenu fluorescenciju gfp izraz su pokrenuli P23 i P20. Međutim, zelena fluorescencija nije primijećena na stanicama pri ekspresiji gfp je bio pod kontrolom P17, P18 i P29 (slika 4). Rezultati konfokalnog mikroskopa su se dobro poklapali sa rezultatima mjerenja intenziteta GFP fluorescencije.

Karakterizacija odabranih promotera i lac promotor putem konfokalnog mikroskopa. (A) Zelena fluorescencija unutar ćelije. (B) Obris ćelijske membrane po mrlji sa FM4-64/L. (C) A i B spojeni zajedno. Sve slike su snimljene pod istim uslovima ekspozicije.

U prethodnoj studiji, skup sintetičkih promotora, koji je sposoban za stabilnu i konstitutivnu ekspresiju nizvodnih gena, primijenjen je za kalibriranu heterolognu ekspresiju gena u P. putida KT2440 pomoću mini-Tn7 dostavnog vektora transposona koji ubacuje promotore u genom P. putida 28 . U drugoj studiji, različite inducibilne promotore karakteriše konstrukcija ProUSER-reporter vektora za upotrebu u P. putida KT2440 i proizvodnja str-u kumarnoj kiselini P. putida KT2440 je poboljšan upotrebom odabranih inducibilnih promotora za optimizaciju ekspresije puta 29 . U ovom radu identificirano je pet snažnih endogenih promotora P4, P6, P9, P16 i P25 iz P. mendocina NK-01 koristeći cevovod koji se sastoji od RNA-seq analize i mjerenja nivoa transkripcije i intenziteta fluorescencije reporterskog gena gfp. Do sada se vrlo malo zna o skriningu jakih promotora iz roda Pseudomonas koristeći strategiju zasnovanu na RNA-seq.

Poboljšana proizvodnja PHA prekomjernom ekspresijom phaC koristeći snažne promotore u P. mendocina NKU

Mcl-PHA sintetički operon od P. mendocina NK-01 je bio izložen u ranijim istraživanjima. Naše istraživanje je pokazalo da PhaC1 najviše doprinosi sintezi mcl-PHA u P. mendocina NK-01 23. Posljedično, prekomjerna ekspresija gena PHA sintaze, posebno phaC1 gen, može imati pozitivan utjecaj na akumulaciju mcl-PHA. Standardna evropska baza podataka vektorske arhitekture (SEVA) razvila je seriju plazmidnih vektora za metabolički inženjering i sintetičku biologiju u Pseudomonas i druge gram-negativne bakterije 30,31. Međutim, ekspresijski sistemi plazmida imaju tendenciju opterećenja bakterija, posebno kada je potrebno više gena za koekspresiju u bakteriji. U ovom su radu 3 endogena snažna promotora P4, P6 i P16 odabrana za prekomjernu ekspresiju gena PHA sintaze neoznačenim umetanjem promotora uzvodno od phaC1 gen u genomu P. mendocina NKU. Ovaj proces nije ostavio nikakve redundantne sekvence u genomu osim umetnutih promotorskih sekvenci (slika 5). Ova strategija uređivanja genoma bez ožiljaka može dati neke prednosti nad prekomjernom ekspresijom plazmida phaC geni.

Konstrukcijski šematski dijagram za umetanje promotora u uzvodno od phaC1 gena i za nokaut phaZ u genomu P. mendocina NKU.

Kromosomskim umetanjem 3 endogena snažna promotora P4, P6 i P16, transkripcijski nivoi phaC1 i phaC2 u rekombinantnim sojevima NKU-4C1, NKU-6C1 i NKU-16C1 poboljšani su u odnosu na soj NKU. posebno, phaC1 pokazao je očiglednije poboljšanje od phaC2 (Slika 6). To može biti posljedica činjenice da phaC1 je bliže umetnutim promotorima nego phaC2.

qPCR analiza i rezultati PHA fermentacije za sojeve NKU-4C1, NKU-6C1, NKU-16C1 i NKU. Transkripcijski nivoi phaC1 (a), phaC2 (b) i phaZ (c) za različite sojeve. (d) Suha težina ćelija (CDW) i proizvodnja PHA za sojeve. Uzorci za qPCR uzeti su nakon 36 h fermentacije PHA. Nivo transkripcije za soj NKU postavljen je na 1. tež.% Definiran je kao omjer PHA i CDW. Podaci predstavljaju srednje vrijednosti ± standardne devijacije trostrukih mjerenja iz tri nezavisna eksperimenta. Studentski t-test je izveden između NKU -a i mutanata. * i ** označavaju P & lt 0,05 i P & lt 0,01, respektivno.

Štaviše, phaZ nalazi između phaC1 i phaC2 imali visoke nivoe transkripcije u rekombinantnim sojevima. Posebno za NKU-6C1 i NKU-16C1, nivoi transkripcije phaZ poboljšane su čak i više phaC1, iako phaZ bio je daleko od promotora u genskoj grupi u odnosu na phaC1. To bi moglo postojati zbog toga što u ovom soju može postojati uska regulatorna sprega između aktivnosti PHA polimeraze i aktivnosti depolimmeraze. Prijavljeno je da pojedinačna prekomjerna ekspresija PhaC -a može dovesti do povećanja ekspresije PhaZ 32. Dakle phaZ pokazala viši nivo transkripcije od phaC1, kada su PhaC i PhaZ istovremeno bili prekomjerno eksprimirani u genskoj grupi. Rezultati PHA fermentacije su pokazali da su svi titri PHA NKU-4C1, NKU-6C1 i NKU-16C1 smanjeni, posebno u NKU-6C1 (slika 6). Ova zapažanja sugeriraju da je prekomjerna ekspresija phaZ može dovesti do prekomjerne sinteze PHA depolimmeraze, te da se unutarstanično akumulirana PHA može razgraditi depolimerazom. Regulatorne uloge PHA depolimmeraze u sintezi PHA prethodno su istraživali drugi istraživači. Na primjer, prekomjerna ekspresija samo PhaC2 u P. putida soj U nije mogao akumulirati veće količine PHA nego u soju divljeg tipa, kao rezultat povišene depolimerizacije PHA u kasnoj fazi sinteze PHA. A phaZ-aktivan mutant P. putida soj U, međutim, akumulirao je više razine PHA od roditeljskog soja 32 . Sadržaj mcl-PHA u a phaZ nokaut mutant of P. putida KT2442 (86 tež.%, Odnos PHA prema CDW) bio je veći od onog kod divljeg soja (66 tež.%) Kada se koristi natrijum oktanoat kao izvor ugljenika 33. Međutim, uklanjanje aktivnosti PHA depolimmeraze u P. putida KT2440 je imao mali uticaj na ukupan prinos PHA 34 . Oba a phaZ-deficitarni mutant P. oleovorans GPo1 35 i dva transpozona poremećeni phaZ mutanti of P. resinovorans 36 nije pokazalo značajno povećanje titra PHA pod različitim uslovima sinteze PHA.

U ovom radu pokušavamo da poboljšamo prinos PHA izgradnjom phaZ nokautni mutanti. Međutim, PHA titar soja NKU-phaZ je smanjen za 4 tež.% u odnosu na soj NKU sa 21 na 17 tež.% (slika 7), što ukazuje da je nokaut phaZ ne može poboljšati prinos i molekularnu težinu mcl-PHA in P. mendocina NK-01. Iznenađujuće, PHA sintetiziraju svi phaZ nokautni mutanti imali su nižu molekularnu težinu od PHA koju je sintetizirao matični soj NKU, s izuzetkom NKU-phaZ-6C1 (Tabela S2). mcl-PHA sintetizirao P. mendocina NKU i njegovi mutantni sojevi uglavnom su bili sastavljeni od tri različita monomera, tj. 3-hidroksioktanoata, 3-hidroksidekanoata i 3-hidroksidodekanoata, kao što je prikazano GC-MS analizom (slike S4, S5). Omjeri sastava monomera mcl-PHA nisu imali očigledne promjene za mutante u poređenju sa NKU (Tabela S3).

qPCR analiza i rezultati PHA fermentacije za sojeve NKU-∆phaZ-4C1, NKU-∆phaZ-6C1, NKU-∆phaZ-16C1 i NKU-∆phaZ. Transkripcijski nivoi phaC1 (a), phaC2 (b) i phaZ (c) za različite sojeve. (d) CDW i PHA proizvodnja za sojeve. Uzorci za qPCR uzeti su nakon 36 h fermentacije PHA. Razina transkripcije za soj NKU-∆phaZ postavljen je kao 1. Podaci predstavljaju srednje vrijednosti ± standardne devijacije trostrukih mjerenja iz tri nezavisna eksperimenta. Studentski t-test je obavljen između NKU-∆phaZ i drugi mutanti. * i ** označavaju P & lt 0,05 i P & lt 0,01, respektivno.

Relativne transkripcijske vrijednosti phaC1 i phaC2 u soju NKU-phaZ-4C1, NKU-phaZ-6C1 i NKU-phaZ-16C1 su svi poboljšani u odnosu na NKU-phaZ, (Slika 7). Zanimljivo je da su relativne transkripcijske vrijednosti phaC1 i phaC2 u soju NKU-phaZ-16C1 su bili najniži među tri navedena soja, dok je PHA titar soja NKU-phaZ-16C1 je bio najveći među gornja tri soja. PHA titar soja NKU-phaZ-4C1 bio je sličan onom soja NKU-phaZ. U usporedbi sa sojem NKU-phaZ, PHA titar soja NKU-phaZ-6C1 je smanjen za 7%, a PHA titar za soj NKU-phaZ-16C1 su poboljšane za 6% na 23 tež.% (Slika 7). Ovi rezultati ukazuju da je nivo ekspresije phaC nije bio pozitivno povezan sa titrom PHA u soju NK-01. U budućim studijama, optimalno izražavanje phaC može biti potrebno za postizanje najvećeg prinosa PHA u soju NK-01. Treba napomenuti da je izvorna RBS sekvenca phaC gen je bio nepromijenjen kada su jaki endogeni promotori ubačeni u uzvodno od phaC operon u genomu P. mendocina. U P. mendocina, nativna RBS sekvenca može biti optimalna za inicijaciju translacije phaC operon. RBS može poslužiti kao važan regulatorni element za inicijaciju translacije i tako očigledno uticati na nivo ekspresije gena 37,38 . Samo upotrebom jakih promotora ne možete postići optimalne nivoe ekspresije. Za ovu studiju, optimizacija RBS sekvence zajedno sa skriningom endogenih jakih promotora može biti potrebna za optimalnu ekspresiju gena PHA sintaze.

Relativne transkripcijske vrijednosti phaC1 i phaC2 za različite mutantne sojeve na 12 h i 24 h mcl-PHA fermentacije su također mjereni, respektivno. Očekivano, sojevi NKU-phaZ-4C1, NKU-phaZ-6C1 i NKU-phaZ-16C1 je pokazao više nivoe transkripcije za phaC1 i phaC2 nego NKU-phaZ u 12 sati, međutim, nisu primijećene značajne razlike među gornja tri soja (slika S6). Nakon 24 sata relativne transkripcijske vrijednosti phaC1 jer su gornja tri soja također poboljšana u usporedbi s NKU-phaZ, i NKU-phaZ-4C1 je imao najviši nivo transkripcije među tri soja (slika S6). Iznenađujuće, nivoi transkripcije za phaC2 u 24 h nije imao očigledan porast za NKU-phaZ-4C1, NKU-phaZ-6C1 i NKU-phaZ-16C1 u usporedbi s NKU-phaZ (Sl. S6). Soj NKU-phaZ-16C1 je pokazao najniže nivoe transkripcije za phaC1 i phaC2 u bilo kojoj vremenskoj tački (Sl. 7 i Sl. S6). Ovi rezultati ukazuju na to da su nivoi transkripcije phaC1 i phaC2 jer mutantni sojevi nisu bili konstantni tokom PHA fermentacije. Promene u nivoima transkripcije gena PHA sintaze tokom perioda fermentacije PHA mogu doprineti manjem povećanju prinosa PHA.

Budući da je složen metabolički put uključen u sintezu PHA iz glukoze, postoje mnogi važni faktori koji utječu na efikasnost sinteze PHA, uključujući Entner-Doudoroffov put, protok acetil-CoA, masne kiseline de novo put sinteze i dostupnost PHA sinteze 39,40,41. Izmjena nekoliko faktora možda neće imati očigledan utjecaj na poboljšanje sinteze PHA.

U ovom radu svi phaZ nokaut mutanti su pokazali smanjenje relativnih nivoa transkripcije phaC1 i phaC2 u poređenju sa njihovim odgovarajućim sojevima bez brisanja phaZ. U usporedbi sa sojem NKU-phaZ, smanjenje titra PHA primijećeno je kod soja NKU-phaZ-4C1 i NKU-phaZ-6C1, dok je PHA titar poboljšan za soj NKU-phaZ-16C1 (slika 7). Ovi rezultati sugeriraju da PhaZ ne samo da je uključen u razgradnju PHA, već također djeluje kao važna uloga u sintezi PHA u P. mendocina NK-01. Prethodna studija pokazala je da PhaZ može odigrati ključnu ulogu u prometu mcl-PHA u uvjetima gladovanja u P. putida KT2442 42. Racionalno podešavanje transkripcijske aktivnosti PHA sintaze i depolimeraze bio bi izvediv pristup za optimizaciju proizvodnje PHA u soju NK-01. Stoga vjerujemo da skrinirani endogeni snažni promotori imaju potencijal primijeniti se za prekomjernu ekspresiju gena na putu sinteze PHA za poboljšanje proizvodnje PHA u P. mendocina NK-01.

Inženjering promotora, kao što je skrining jakih promotora, široko se primjenjuje za inženjering metaboličkih puteva kako bi se poboljšao prinos mnogih industrijskih proizvoda. Međutim, u mnogim slučajevima, egzogeni promotori možda nisu kompatibilni sa sistemima ekspresije nativnih gena u P. mendocina NK-01. Prethodno istraživanje u našoj laboratoriji pokazalo je da je prinos PHA imao očigledan pad nakon prekomjerne ekspresije gena PHA sintaze korištenjem egzogenog snažnog promotora J23119 u P. putida KT2440 (neobjavljeni podaci). Ovo je također pokazalo da nije da što je veća ekspresija PhaC-a, veći je prinos mcl-PHA mogao biti. U ovoj studiji nismo odabrali jako jak egzogeni promotor kao referentni i često korišteni promotor lac promotor 43,44 je korišten kao kontrola u testovima transkripcijske aktivnosti za skrining odgovarajućih endogenih snažnih promotora. U poređenju sa lac promoter, skrinirani endogeni promotori P4, P6 i P16 pokazali su veću transkripcionu aktivnost i intenzitet fluorescencije. Stoga smo testirali sposobnost pregledanih endogenih promotora da poboljšaju proizvodnju mcl-PHA prekomjernom ekspresijom PHA sintaze u P. mendocina NK-01. Potreban je budući rad kako bi se provjerili prikladniji promotori i optimizirao biosintetski put PHA kako bi se dodatno poboljšala proizvodnja mcl-PHA, ne samo prekomernom ekspresijom gena PHA sintaze. A provjereni endogeni promotori se također mogu primijeniti za poboljšanje biosinteze AO, što je još jedan proizvod koji sintetizira NK-01 iz glukoze. Upotreba endogenih promotora može biti izvodljiva metoda za optimizaciju ekspresije gena sintetičkog puta, a ova strategija bi se potencijalno mogla iskoristiti za pojačanu proizvodnju drugih vrijednih bioloških proizvoda.


Sadržaj

Dva najčešće korišćena inducibilna ekspresiona sistema za istraživanje biologije eukariotske ćelije nazivaju se Tet-Off i Tet-On. [3] Tet-Off sistem za kontrolu ekspresije gena od interesa u ćelijama sisara razvili su profesori Hermann Bujard i Manfred Gossen na Univerzitetu u Heidelbergu i prvi put objavljen 1992. [4]

Tet-Off sistem koristi tetraciklinski transaktivator (tTA) protein, koji nastaje spajanjem jednog proteina, TetR (tetraciklinski represor), koji se nalazi u Escherichia coli bakterije, sa domenom aktivacije drugog proteina, VP16, koji se nalazi u virusu Herpes Simplex. [5]

Rezultirajući tTA protein se može specifično vezati za DNK TetO operatorske sekvence. U većini Tet-Off sistema, nekoliko ponavljanja takvih TetO sekvenci postavljeno je uzvodno od minimalnog promotora kao što je CMV promotor. Cjelovitost nekoliko TetO sekvenci s minimalnim promotorom naziva se a element odgovora tetraciklina (TRE), jer reagira na vezivanje proteina tetraciklinskog transaktivatora tTA povećanom ekspresijom gena ili gena nizvodno od njegovog promotora.

U Tet-Off sistemu, ekspresija TRE kontrolisanih gena može biti potisnuta tetraciklinom i njegovim derivatima. Oni vezuju tTA i čine je nesposobnom za vezivanje za TRE sekvence, čime se sprječava transaktivacija TRE kontroliranih gena.

Tet-On sistem radi slično, ali na suprotan način. Dok je u Tet-Off sistemu, tTA može vezati operatora samo ako ne vezan za tetraciklin ili jedan od njegovih derivata, poput doksiciklina, u Tet-On sistemu, protein rtTA je sposoban da veže operatora samo ako vezani tetraciklinom. Tako uvođenje doksiciklina u sistem pokreće transkripciju genetskog proizvoda. Tet-On sistem se ponekad preferira u odnosu na Tet-Off zbog bržeg odziva.

Tet-Off sistemi ekspresije se takođe koriste u generisanju transgenih miševa koji uslovno izražavaju gen od interesa.

Tet-On Advanced transaktivator (takođe poznat kao rtTA2 S -M2) je alternativna verzija Tet-On-a koja pokazuje smanjenu bazalnu ekspresiju i funkcionira pri 10 puta nižoj koncentraciji Doxa od Tet-Off-a. Osim toga, smatra se da je njegova ekspresija stabilnija u eukariotskim stanicama jer je optimiziran za ljudski kodon i koristi 3 minimalne domene transkripcijske aktivacije. Otkriven je 2000. godine kao jedan od dva poboljšana mutanta od strane H. Bujarda i njegovih kolega nakon nasumične mutageneze Tet Repressor dijela gena transaktivatora. [6] Tet-On 3G (također poznat kao rtTA-V10 [7] ) je sličan Tet-On Advanced, ali je izveden iz rtTA2 S -S2 umjesto rtTA2 S -M2. Također je optimiziran za ljudski kodon i sastoji se od 3 minimalne domene aktivacije VP16. Međutim, Tet-On 3G protein ima 5 aminokiselinskih razlika u odnosu na Tet-On Advanced koje izgleda da dodatno povećavaju njegovu osjetljivost na Dox. Tet-On 3G je osjetljiv na 100 puta manje Doxa i 7 je puta aktivniji od originalnog Tet-On-a. [8]

Drugi sistemi kao što je T-REx sistem kompanije Life Technologies rade na drugačiji način. [9] Gen od interesa je okružen uzvodnim CMV promotorom i dva TetO2 mjesta. Ekspresija gena od interesa je potisnuta visokim afinitetom vezivanja TetR homodimera za svaku TetO2 sekvencu u odsustvu tetraciklina. Uvođenje tetraciklina dovodi do vezivanja jednog tetraciklina na svakom TetR homodimeru, nakon čega slijedi oslobađanje TetO2 od TetR homodimera. Odvajanje TetR homodimera i TetO2 dovodi do derepresije gena od interesa.

Modificirana verzija T-REx-a je Linearizer sintetički biološki krug, optimiziran za podešavanje ekspresije gena u eukariotskim (kvasac za pupanje, ljudski, itd.) stanicama. By incorporating TetO2 sites into the promoter driving TetR expression, it creates negative feedback, which ensures homogeneous expression (low noise) and a linear dose-response to tetracycline analogs. [10]

In the most commonly used plasmids, the tetracycline response element consists of 7 repeats of the 19bp bacterial TetO sequence ( TCCCTATCAGTGATAGAGA ) separated by spacer sequences (for example: ACGATGTCGAGTTTAC ). It is the TetO that is recognized and bound by the TetR portion of Tet-On or Tet-Off. The TRE is usually placed upstream of a minimal promoter that has very low basal expression in the absence of bound Tet-Off (or Tet-On).

The Tet system has advantages over Cre, FRT, and ER (estrogen receptor) conditional gene expression systems. In the Cre and FRT systems, activation or knockout of the gene is irreversible once recombination is accomplished, whereas, in Tet and ER systems, it is reversible. The Tet system has very tight control on expression, whereas ER system is somewhat leaky. [11] However, the Tet system, which depends on transcription and subsequent translation of a target gene, is not as fast-acting as the ER system, which stabilizes the already-expressed target protein upon hormone administration. Also, since the 19bp tet-o sequence is naturally absent from mammalian cells, pleiotropy is thought to be minimized compared to hormonal methods of control. When using the Tet system in cell culture, it is important to confirm that each batch of fetal bovine serum is tested to confirm that contaminating tetracyclines are absent or are too low to interfere with inducibility.

The mechanism of action for the antibacterial effect of tetracyclines relies on disrupting protein translation in bacteria, thereby damaging the ability of microbes to grow and repair however protein translation is also disrupted in eukaryotic mitochondria leading to effects that may confound experimental results. [12] [13]


Metode

Chick embryos

Fertilized chick eggs from a commercial source (JA57 strain, Dangers, France) were incubated at 38.5 °C. Embryos were staged according to days in ovo. For early stages, the following day numbers and HH (Hamburger and Hamilton) stages [56] are equivalent: E2/HH13, E2.5/HH15 and correspond to 20 and 25 somite stages, respectively.

Establishment of recombinant vectors

The pT2AL-MLC-Tomato-T2A-GFP plasmid was obtained as following: The Myr-TdTomato-T2A sequence was amplified by PCR from the plasmid pCS2-TdTomato-2A-GFP [52]. To facilitate subsequent cloning, one XhoI site was added to the forward primer and one BstBI site was added to the reverse primer. The purified PCR product was then inserted into pCRII-TOPO (Invitrogen) and a clone with Tomato downstream of SP6 promoter was selected, giving rise to a plasmid named TOPO/Tomato. H2B-GFP was amplified by PCR from the plasmid pCS2-TdTomato-2A-GFP [52]. A BstbI site was added to the forward primer and one PmlI site and one ClaI site were added to the reverse primer. The purified PCR product was then inserted into pCRII-TOPO (Invitrogen) and a clone with GFP downstream of SP6 promoter was selected, resulting in a plasmid called TOPO/GFP. Next, both TOPO/Tomato and TOPO/GFP were digested with BstbI and NotI. The T2A sequence was then inserted into TOPO/Tomato using the T4 DNA ligase (New England Biolabs) to generate a plasmid named TOPO/Tomato-T2A-GFP. The Tomato-T2A-GFP cassette was then excised from TOPO Tomato-T2A-GFP using EcoRV and XhoI and cloned into the pT2AL200R150G [57] previously digested with ClaI (blunt-ended using Fermentas T4 DNA polymerase) and XhoI. The resulting plasmid was named pT2AL-Tomato-T2A-GFP. The Myosin Light Chain (MLC) mouse promoter was removed from the pT2K-MLC-Fgf4 plasmid (previously described in [44]) using NcoI and XhoI. Both extremities were then blunt-ended using T4 DNA polymerase (Fermentas). The MLC promoter was next blunt ligated to TOPO GFP previously digested with XbaI made blunt. A clone with the MLC promoter inserted with ApaI in 5’ and XhoI in 3’ was selected resulting in a plasmid called TOPO/GFP/MLC. Both TOPO/GFP/MLC and pT2AL-Tomato-T2A-GFP were digested with ApaI and XhoI. MLC was inserted into pT2AL-Tomato-T2A-GFP to obtain pT2AL-MLC-Tomato-T2A-GFP.

The pT2AL-CMV/βactin-Tomato-T2A-GFP plasmid was obtained as followed: The pT2AL-MLC-Tomato-T2A-GFP plasmid was digested with ApaI (blunt-ended using Fermentas T4 DNA polymerase) and SphI to remove the MLC promoter. The MLC promoter was then replaced by the CMV-βactin promoter (the chick βactin promoter downstream of a CMV enhancer), which was excised from the CMV-βactin-EGFP [35] using SalI (blunt-ended) and SphI to generate the pT2AL-CMV-Tomato-T2A-GFP plasmid.

The pT2AL-p57/βactin -Tomato-T2A-GFP plasmid was obtained as followed: The p57MRE regulatory sequence was excised from pSK-p57MRE plasmid [11] by digestion with Spe1 and SacII. The CMV enhancer of the pT2AL-CMV/βactin-Tomato-T2A-GFP plasmid was excised by Acc1 and SnaBI and replaced with the p57MRE using blunt ligation with the Rapid DNA Ligation Kit (Roche). The generated plasmid was named the pT2AL-p57/βactin-Tomato-T2A-GFP.

Electroporation

Limb somite electroporation was performed as previously described [35]. The DNA solution was systematically composed of the Tol2 stable vectors and the transient transposase vector CMV/βactin-T2TP, which allows the stable integration into the chick genome. The concentration of the different vectors was between 1.5 and 2 μg/μL and of 1/3 for the CMV/βactin-T2TP. DNA was prepared in solution containing carboxymethyl cellulose 0,17 %, fast green 1 %, MgCl2 1 mM and PBS 1X in water.

Lateral plate electroporation was performed as followed: Stage HH13–15 (E2) chick embryos were windowed following standard techniques in preparation for electroporation [58]. PBS without Ca 2+ /Mg 2+ was applied to the embryo. A capillary was backfilled with DNA solution, which was injected under 200 Pa pressure (injection duration 0.1–0.5 s and compensatory pressure 15–25 Pa) (Femtojet, Eppendorf) into the embryonic coelom, to fill completely the anterior to posterior extent of the forelimb territory. The negative electrode (0.8 mm diameter tungsten rod with a 4-mm length and 2-mm exposed surface) was inserted into the yolk and positioned beneath the forelimb field, approximately 2 mm below the embryo. A 0.8 mm diameter platinum rod with a 1-mm exposed tip served as the positive electrode and was positioned above the forelimb field with an approximate distance of 3 mm. A wave pulse train consisting of 50 V, five pulses, 20 ms duration with a 200 ms interpulse interval was delivered via TSS20 electroporator and EP21 current amplifier (Intracel). Embryos were returned to 37.5 °C for the remaining incubation period. DNA solution was composed of pT2AL-CMV/βactin-Tomato-T2A-GFP (1-3 μg/μL) and CMV/βactin-T2TP at a molar ratio of 1:5–1:10, diluted in a mix containing PBS without Ca 2+ /Mg 2+ and Fast Green 0.005 %. This ratio resulted in persistent gene expression in the embryonic limbs during foetal development.

Imunohistokemija

Experimental forelimbs were fixed in paraformaldehyde 4 % overnight at 4 °C and processed for cryostat sections (12 μm). Immunohistochemistry was performed as previously described [59]. The monoclonal antibodies MF20 that recognizes sarcomeric myosin heavy chains and Pax7 that recognizes muscle progenitors, developed by D.A. Fischman and A. Kawakami, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology Iowa City, IA 52242. After overnight incubation with the primary antibody at 4 °C, biotinylated secondary antibodies (Anti-Mouse IgG2b from Southern Biotech Anti-Mouse IgG1 from Jackson ImmunoResearch laboratories) were applied for 1 h at room temperature, followed by a 45 min incubation with Cy5-Streptavidin (Invitrogen). Hoechst (Molecular Probes) staining was performed with a dilution of 1/20000 in PBS 1X for 10 min at room temperature.

In situ hibridizacija

In situ hybridization experiments were performed for GFP i Scx probes, as previously described [35].

Image capturing

Images of the wholemount electroporated limbs were acquired with a Leica stereo-macroscope equipped with a Leica DFC300 camera. After immunohistochemistry, sectioned samples images were captured using a Nikon epifluorescence microscope, a Leica DMI600B inverted microscope or a Leica SP5 confocal system.


MATERIJALI I METODE

ASC Isolation and Clonal Culture

Stromal vascular cells with a CD34 + CD105 + CD45 − CD31 − phenotype (ASCs) were isolated from human adipose tissue (Boquest et al, 2005.). In short, tissue was obtained by liposuction from the hip and thigh regions of healthy women. After washing in Hank’s balanced salt solution (HBSS), the tissue was digested for 2 h at 37°C in HBSS with collagenase and DNase I. Adipocytes were separated from stromal vascular cells after sedimentation at 400 × g for 10 min and removed by aspiration. Erythrocytes were removed by resuspending stromal vascular cell pellets in lysis buffer (2.06 mg/ml Tris base, pH 7.2, and 7.49 mg/ml NH4Cl) for 10 min. After centrifugation, pellets were resuspended in HBSS containing 2% fetal bovine serum (FBS) (Sigma-Aldrich. St. Louis, MO) and passed through a 100-μm sieve and a 40-μm sieve. CD45 + cells were removed with paramagnetic beads conjugated to mouse anti-human CD45 monoclonal antibodies (Miltenyi Biotech, Bergish Gladbach, Germany) using a superMACS magnet (Miltenyi Biotech). Remaining CD45 − cells were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD31 antibodies (Serotec, Oxford, United Kingdom) at a concentration of 10 μl of antibody per 10 6 cells for 15 min at 4°C. Cells were washed and incubated with anti-FITC microbeads (Miltenyi Biotech) for 15 min at 4°C. CD31 − and CD31 + cells were separated using an LS column (Miltenyi Biotech). CD31 − cells were reexposed to a new LS column to eliminate any leftover contaminating CD31 + cells. Flow cytometry analysis of each cell subset from each donor indicated that purity was >98% (our unpublished data) (Boquest et al, 2005.). Aliquots of each cell subset were immediately snap-frozen in liquid nitrogen for DNA and RNA isolations, or they were cultured.

CD31 − clonal cell lines were generated by culturing single CD31 − cells in each well of 48-well plates in DMEM/F-12 medium containing 50% FBS and antibiotics. After ∼16 h, the medium was replaced by DMEM/F-12 with 20% FBS. After ∼1 wk, colonies containing >10 cells were passaged by trypsinization and expanded. Only clonal lines that could be easily expanded were used in this study. Clones A1 and A2, and clones B1, B2, and B3 examined in this study were from two different female donors (age 27 and 39, respectively).

Adipogenic Differentiation

Clonal ASC lines generated from individual CD31 − cells at passage 4 were cultured to confluence before differentiation. For adipogenic differentiation (Zuk et al., 2001), cells cultured in DMEM/F-12 with 10% FBS were stimulated for 3 wk with 0.5 mM 1-methyl-3 isobutylxanthine, 1 μM dexamethasone, 10 μg/ml insulin (Novo Nordisk, Copenhagen, Denmark), and 200 μM indomethacin (Dumex-Alpharma, Copenhagen, Denmark). To visualize lipid droplets, formalin-fixed cells were washed in 50% isopropanol and stained with Oil Red-O.

Gene Loci and Regions Analyzed by Bisulfite Sequencing

Supplemental Figure S1 illustrates the promoter regions of the genes analyzed by bisulfite sequencing in this study. We examined four adipogenic genes, including leptin (LEP) (Mason et al., 1998 Reseland et al., 2001), peroxisome proliferator-activated receptor gamma 2 (PPARG2) (Fajas et al., 1997), fatty acid-binding protein 4 (FABP4) (Ross et al., 1990 Graves et al., 1992), and lipoprotein lipase (LPL) (Bey et al., 1998 Merkel et al, 2002.). We also examined genes unrelated to adipogenesis, such as myogenin (MYOG), a basic helix-loop-helix transcription factor required for myocyte differentiation (Massari and Murre, 2000) the endothelial marker gene CD31/PCAM-1 (Cao et al., 2002 Chi et al., 2003) and the constitutively expressed housekeeping gene GAPDH. The LEP promoter region analyzed was from nucleotides 2719–2937 (GenBank accession no. U43589) and spanned 27 potentially methylated cytosines in CpG dinucleotides starting 42 base pairs upstream of the ATG translational start site. The LEP proximal promoter activity is known to be regulated by DNA methylation (Melzner et al, 2002.). The PPARG2 promoter region (Fajas et al., 1997) spanned nucleotides 108–587 (GenBank accession no. AB005520) and included 6 CpGs starting 264 base pairs upstream of the ATG. The FABP4 (GenBank accession no. NM_001442) promoter region examined was identified using ENSEMBL and encompassed four CpGs starting 130 base pairs upstream of the ATG. The LPL promoter region spanned bases 1321–1777 (GenBank accession no. X68111) and included 11 CpGs starting 134 base pairs upstream of the ATG. The MYOG region analyzed spanned nucleotides 1268–1484 (GenBank accession no. X62155) and included 16 CpGs starting 87 base pairs downstream of the ATG. The CD31 promoter region examined included nucleotides 1095–1480 (GenBank accession no. X96848) and included 18 CpGs ranging from nucleotide −352 to +34 relative to the ATG. The GAPDH promoter region spanned bases 1121–1337 (GenBank accession no. J04038) and encompassed 28 CpGs 116 base pairs upstream of the ATG.

Bisulfite Sequencing

DNA was purified either using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich), or for most samples, by phenol-chloroform-isoamyl alcohol extraction. In the latter case, cells were first lysed for 10 min in lysis buffer (10 mM Tris-HCl, pH 8, 100 mM EDTA, and 0.5% SDS) and digested with 0.1 mg/ml proteinase K overnight. Bisulfite conversion (Warnecke et al., 2002) was performed using the MethylEasy DNA bisulfite modification kit (Human Genetic Signatures, Sydney, Australia). Converted DNA was used fresh or stored at −20°C. Converted DNA was amplified by PCR using primer sets purchased from Human Genetic Signatures for the LEP, MYOG, CD31 i GAPDH geni. These primers sets are commercially available (www.geneticsignatures.com). We also designed primers using the Methprimer software (www.urogene.org/methprimer/index1.html) for the PPARG2, FABP4, i LPL genes (Table 1). Za PPARG2, FABP4, i LPL, PCR conditions were 95°C for 7 min and 40 cycles of 95°C 1 min, 54°C 2 min and 72°C 2 min, followed by 10 min at 72°C. Za LEP, MYOG, CD31, i GAPDH, nested PCRs were performed, each as follows: 95°C for 3 min and 30 cycles of 95°C for 1 min, 50°C for 2 min, and 72°C for 2 min, followed by 10 min at 72°C. PCR products were directly sequenced or cloned into bacteria using the TOPO TA cloning kit (Invitrogen, Oslo, Norway). Clones were sequenced using commercial services from MWG Biotech (Ebersberg, Germany).

Table 1. Bisulfite sequencing primers used in this study

a Purchased from Human Genetic Signatures.

Real-Time Reverse Transcription (RT)-PCR

RT-PCR was carried from 500 ng of total RNA using the Iscript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative (Q)RT-PCR reactions were performed in triplicates on a MyiQ real-time PCR Detection System using IQ SYBR Green (Bio-Rad). Most samples were analyzed in duplicates from two separate cDNA preparations. Primers used are listed in Table 2. SYBR Green PCR conditions were 95°C for 4.5 min and 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, using GAPDH as a normalization control. mRNA levels were calculated as described previously (Pfaffl, 2001).

Table 2. Real-time RT-PCR primers used in this study


Expression Vectors: Types & Characteristics

The expression vectors are vectors which act as vehicles for DNA insert and also allow the DNA insert to be expressed efficiently. These may be plasmids or viruses. The expression vectors are also known as expression constructs.

The expression vectors are genetically engineered for the introduction of genes into the target cells. In addition to the gene of interest, these expression constructs also contain regulatory elements like enhancers and promoters so that efficient transcription of the gene of interest occurs.

The simplest expression constructs are also known as transcription vectors only because they allow transcription of the cloned foreign gene and not its translation. The vectors which facilitate both transcription and translation of the cloned foreign gene are known as protein expression vectors. These protein expression constructs also lead to the production of recombinant protein.

Now, for transcription and translation, a promoter and a termination sequence are a must. Transcription initiates at the promoter and ends at the termination site. The promoters of expression vectors must have on/off switches. These switches help in the regulation of production of the gene product. Excessive amounts of product of the gene of interest can be toxic for the cell. A common promoter utilized in the expression constructs is the mutant version of the lac promoter, lacUV. The lacUV promoter initiates a high level of transcription under induced conditions. Moreover, in some expression vectors, a ribosomal binding site is present upstream to the start codon. The ribosomal binding site facilitates the efficient translation of the cloned foreign gene.

Expression vectors are used extensively in molecular biology in techniques like site-directed mutagenesis.

How do Expression Vectors work?

  • Once the expression construct is inside the host cell, the protein encoded by the gene of interest is produced by the transcription. Thereafter, it utilizes the translation machinery and ribosomal complexes of the host organism.
  • Frequently, the plasmid is genetically engineered to harbor regulatory elements like enhancers and promoters. These regulator sequences aid in efficient transcription of the gene of interest.
  • Expression vectors are extensively used as tools which help in the production of mRNAs and, in turn, stable proteins. They are of much interest in biotechnology and molecular biology for the production of proteins like insulin. Insulin is the chief ingredient in the treatment of the complex disease, Diabetes.
  • When the protein product is expressed, it is to be then purified. The purification of a protein poses a challenge since the protein of interest, whose gene is carried on the expression vector, is to be purified independently of the proteins of the host organism. To make the process of purification simpler, the gene of interest carried on the expression vector should always have a ‘tag’. This tag can be any marker peptide or histidine (His tag).
  • Expression vectors are considerably exploited in techniques like site-directed mutagenesis. Cloning vectors introduce the gene of interest into a plasmid which in turn replicates in bacteria. These cloning vectors need not necessarily result in the expression of a protein.

Therefore, expression vectors must have the following expression signals:

  • Strong promoter,
  • Strong termination codon,
  • Adjustment of distance between the promoter and cloned gene,
  • Inserted transcription termination sequence, and
  • Portable translation initiation sequence.

Promoter

  • A promoter ensures a reliable transcription of the gene of interest. Also, strong promoters are also necessary for an efficient mRNA synthesis with RNA polymerase.
  • Regulation of the promoter is another critical aspect which should always be kept in mind while constructing an expression vector.
  • The strongest promoters are those found in bacteriophages T5 and T7.

U E. coli, the promoter is regulated in two ways:

Induction : the addition of chemical switches on the transcription of the gene.

Repression : addition of chemical switches off the transcription of the gene.

The most commonly used promoters in E. coli expression system are:

  • It regulates the transcription of the lac Z gene. The lac Z gene is responsible for the production of β- galactosidase.
  • The lac Z gene can be induced by IPTG, isopropylthiogalactosidase.
  • The lac promoter sequences can be fused to the target gene. It will, then, result in lactose- dependent expression of the target gene.
  • Nevertheless, the lac promoter has its drawbacks. It is quite weak and cannot be utilized for the high levels of production of the desired protein. In addition to this, the lac genes carry out the basal level of transcription even in the absence of induction (inducer molecule).
  • It is responsible for the regulation of a cluster of genes which are involved in tryptophan biosynthesis.
  • Tryptophan acts as its repressor molecule, and it is induced by 3-β-indoleacrylic acid.
  • It is formed by hybridization of the lac and trp promoter. However, it is stronger than either of them.
  • The tac promoter is induced by IPTG, isopropylthiogalactosidase.
  • It is a strong promoter and is responsible for transcription of λDNA in E. coli
  • The product of λcI gene acts as its repressor. It is called λ repressor.
  • The expression construct with the λPL promoter is used in combination with the E. coli mutant host. It is responsible for the production of a temperature sensitive form of λ repressor.
  • At low temperatures, the repressor protein represses the transcription whereas the transcription of the cloned gene occurs at high temperatures because the repressor is inactivated at high temperature.
  • For the expression of proteins in mammalian cells, the promoter must be located upstream of the cloned cDNA for its efficient transcription.
  • In most of the cases, viral promoters are employed only because they are reliable for a strong constitutive expression.
  • The widely used promoters are CMV promoter (derived from cytomegalovirus) and the SV40 promoter (derived from simian virus 40).

The promoters in the commercially available yeast expression vectors may be active constitutively or inducible ones.

A constitutive promoter is a kind of promoter which is unregulated and allows continual transcription of its associated gene.

Example of a constitutive promoter: GAP promoter of the gene encoding glyceraldehyde-3-phosphate dehydrogenase.

An inducible promoter is the one which works in a regulated manner and the expression of genes associated with them can be switched on or off at a particular stage of development or at a certain point of time.

Examples of inducible promoters: AOX1, GAL1, GAL10, nmt1, nmt42, and nmt81.

The AOX1 promoter of the gene encoding alcohol oxidase. It is induced by methanol and is best-suited for expression of the protein in Pichia pastoris.

The GAL1 i GAL10 promoters are other examples. They are induced by galactose and are suitable for protein expression in Saccharomyces cerevisiae.

The nmt1, nmt42, i nmt81 promoters which are induced by thiamine for protein expression in Schizosaccharomyces pombe.

Reporter Gene

  • The reporter gene is responsible for the production of the protein which can be detected and quantified with the help of a simple assay.
  • They serve as a tool to measure the efficiency of the gene expression and also to detect the intracellular localization of the protein.
  • The rate of expression of the structural gene is dependent upon the regulatory sequences which are located upstream to it.
  • The rate of expression of the gene can be measured by replacement of its protein-encoding portion. Also, it can be fused to another gene which expresses another protein. The presence of this another protein can be easily identified.
  • Reporter genes are useful in the identification of promoters, enhancers, and other proteins or regulatory elements which bind to them.

The most commonly utilized reporter genes are:

  • It acts as a reporter in the presence of X- gal.
  • Its levels are easily detected by the intensity of colour which is produced. The intensity of the blue colour produced is quantified.

2. CAT (chloramphenicol acetyltransferase) encoding gene of E. coli

  • The CAT gene encodes chloramphenicol acetyltransferase.
  • The transferase enzyme is responsible for the transfer of acetyl groups from acetyl CoA to the recipient antibiotic, chloramphenicol

3. Luciferase encoding gene of firefly, Photinus pyralis

  • Luciferase is accountable for the oxidation of luciferin.
  • The oxidation of luciferin results in the emission of yellow-green light. The emission of light is easily detected irrespective of the low levels.

4. Green fluorescent protein (GFP) encoding gene of jellyfish, Aequorea victoria

  • GFP was discovered by Shimomura.
  • It is an autofluorescent protein with 238 amino acid residues produced by the bioluminescent jellyfish Aequorea victoria.
  • In GFP, β-barrel is formed by eleven β strands. An α- helix runs through the center. The chromophore is located in the middle of the barrel. The amino acid residues from 65 to 67 with sequence Ser-Tyr-Gly form the chromophore, p- hydroxybenzylideneimidazolinone, which is fluorescent. The chromophore fluoresces at a peak wavelength of 508 nm (green light) when it is irradiated with UV or blue light (400 nm).
  • GFP serves as a tool for determining protein localization.
  • It serves as a tag whereby it is fused with a protein whose expression is to be monitored. Basically, the subcellular localization of the protein is investigated.
  • Genetic engineering techniques help in the production of vectors which contain the coding sequence of the unidentified protein, X, cloned in the coding sequence of the GFP.
  • This fusion product of GFP-X can now be transfected into target cells and the expression, as well as the subcellular location of the X protein, can easily be monitored and detected.

Ribosome Binding Site and Translation Initiation Site

  • The ribosomal binding site (RBS) follows the promoter. It is responsible for the efficient translation of the cloned gene.
  • The translation initiation site in case of prokaryotes is known as the Shine Dalgarno sequence. This sequence is enclosed within the RBS only.
  • The consensus sequence of the translation initiation site includes a set of 8 base pairs present upstream the AUG start codon.
  • The translation in eukaryotes is initiated at a particular sequence called Kozak sequence.
  • The ribosomal machinery for the translation of mRNA is assembled on this site.

Polylinkers

  • Each vector contains particular recognition sites for restriction enzymes. It is at the restriction site that the vector is excised to clone the foreign gene of interest.
  • These sites often lie close together and, hence, are called polylinkers or multiple cloning sites (MCS).
  • These regions are 50 to 100 base pair in length and may have a cluster of up to 25 restriction sites.

Poly-A (polyadenylation) Tail

  • The poly-A tail present, at the end of the mRNA formed, protects the mRNA from degradation by the exonucleases or endonucleases.
  • It is extremely critical for the stability of the mRNA.
  • It is also responsible for the termination of transcription and translation and stabilizes the mRNA production.
  • A nucleolytic enzyme complex and a poly-A-polymerase are prerequisites for the addition of poly-A tail at the end of the mRNA.

Expression System

The production of a protein requires an expression system. There are two types of expression systems, prokaryotic and eukaryotic expression system. Each of them has its own advantages and drawbacks which can be taken into consideration while constructing an expression system. However, there is no such expression system which can be considered universal for the heterologous protein production.

Prokaryotic Expression System

  • The specificity of the promoter of an RNA polymerase, in the case of prokaryotes, is mediated by sigma factor.
  • E. coli is the widely used prokaryotic expression system.
  • It expresses high levels of the protein.
  • The E. coli strains are manipulated genetically for the production of recombinant protein so that they are rendered safe for large-scale experiments and fermentation.
  • The purification of the protein has become easier since recombinant-fusion proteins can be purified by affinity chromatography, say for example glutathione-S-transferase and maltose-binding fusion proteins.

Regardless of the advancements and improvements occurring,in the prokaryotic expression system, there are still many difficulties associated and challenges posed by the production of protein from the cloned foreign genes. These kinds of challenges can be grouped together into 2 categories:


Resilience

Reducing the effects of significant adversity on children’s healthy development is essential to the progress and prosperity of any society. Science tells us that some children develop otpornost, or the ability to overcome serious hardship, while others do not. Understanding why some children do well despite adverse early experiences is crucial, because it can inform more effective policies and programs that help more children reach their full potential.

One way to understand the development of resilience is to visualize a balance scale or seesaw. Protective experiences and coping skills on one side counterbalance significant adversity on the other. Resilience is evident when a child’s health and development tips toward positive outcomes — even when a heavy load of factors is stacked on the negative outcome side.

Over time, the cumulative impact of positive life experiences and coping skills can shift the fulcrum’s position, making it easier to achieve positive outcomes. Play Tipping the Scales: The Resilience Game to learn more.

The single most common factor for children who develop resilience is at least one stable and committed relationship with a supportive parent, caregiver, or other adult. These relationships provide the personalized responsiveness, scaffolding, and protection that buffer children from developmental disruption. They also build key capacities—such as the ability to plan, monitor, and regulate behavior—that enable children to respond adaptively to adversity and thrive. This combination of supportive relationships, adaptive skill-building, and positive experiences is the foundation of resilience.

Children who do well in the face of serious hardship typically have a biological resistance to adversity i strong relationships with the important adults in their family and community. Resilience is the result of a combination of protective factors. Neither individual characteristics nor social environments alone are likely to ensure positive outcomes for children who experience prolonged periods of toxic stress. It is the interaction between biology and environment that builds a child’s ability to cope with adversity and overcome threats to healthy development.

Research has identified a common set of factors that predispose children to positive outcomes in the face of significant adversity. Individuals who demonstrate resilience in response to one form of adversity may not necessarily do so in response to another. Yet when these positive influences are operating effectively, they “stack the scale” with positive weight and optimize resilience across multiple contexts. These counterbalancing factors include

  1. facilitating supportive adult-child relationships
  2. building a sense of self-efficacy and perceived control
  3. providing opportunities to strengthen adaptive skills and self-regulatory capacities and
  4. mobilizing sources of faith, hope, and cultural traditions.

Learning to cope with manageable threats is critical for the development of resilience. Not all stress is harmful. There are numerous opportunities in every child’s life to experience manageable stress—and with the help of supportive adults, this “positive stress” can be growth-promoting. Over time, we become better able to cope with life’s obstacles and hardships, both physically and mentally.

The capabilities that underlie resilience can be strengthened at any age. The brain and other biological systems are most adaptable early in life. Yet while their development lays the foundation for a wide range of resilient behaviors, it is never too late to build resilience. Age-appropriate, health-promoting activities can significantly improve the odds that an individual will recover from stress-inducing experiences. For example, regular physical exercise, stress-reduction practices, and programs that actively build executive function and self-regulation skills can improve the abilities of children and adults to cope with, adapt to, and even prevent adversity in their lives. Adults who strengthen these skills in themselves can better model healthy behaviors for their children, thereby improving the resilience of the next generation.


Podaci o autoru

Present address: Federal Environment Agency, Section IV 2.2 Pharmaceuticals, Washing and Cleansing Agents, and Nanotechnology, Wörlitzer Platz 1, 06844, Dessau, Germany

Pripadnosti

Department of Biochemistry, University of Bayreuth, Universitätsstr. 30, 95447, Bayreuth, Germany

Molecular Biology of Archaea, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Strasse, 35043, Marburg, Germany

Alexander Wlodkowski & Sonja-Verena Albers

Institute of Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences of Northwestern Switzerland (FHNW), Gründenstrasse 40, 4132, Muttenz, Switzerland


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