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Kada je potrebna stroga sterilna tehnika? Kloniranje u odnosu na ekspresiju proteina

Kada je potrebna stroga sterilna tehnika? Kloniranje u odnosu na ekspresiju proteina


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Pozadina: Prije sam radio s RNA, a zatim koristimo laminarne kapuljače za sve radove na kojima obavljamo sadnju ili inokulaciju kultura primjenom sterilne tehnike. Sada radim u biokemijskom laboratoriju. Rečeno mi je da mogu raditi na laboratorijskom stolu, dok obavljam osnovno kloniranje za proizvodnju plazmida u E.coli, i ne moram koristiti Bunsenov plamenik sve dok primjenjujem sterilnu tehniku ​​(a u većini slučajeva ne ne vidim ljude kako steriliraju klupu prije posla). Međutim, ako radim bilo koji posao koji se odnosi na ekspresiju proteina - tada bih trebao koristiti Bunsen -plamenik dok primjenjujem sterilnu tehniku.

Pitanje: Je li zaista tako da čak ni ne morate koristiti Bunsenov plamenik za vrijeme oplate (normalne E.coli: XL1-plave stanice)? u ovom slučaju, zašto? Zar u laboratoriju ne postoji uvijek nešto što može rasti na pločama s kanamicinom? Ne koristimo bijelo/plavo probiranje, jednostavno širimo XL1-plave stanice na LB ploče s kanamicinom-zatim odaberemo koloniju prosječne veličine da napravimo LB-kulturu. Čini mi se da smo samo sretni što obično odaberemo pravu koloniju (što potvrđujemo sekvenciranjem nakon mini-pripreme).


Kratak odgovor je da nikad ne boli vježbati temeljitu aseptičku tehniku.

Moje opće pravilo je da, ako otvaram kulturu bakterija (ploče, eppendorfi, epruvete itd.), To činim pod bunsenom - iznimka je ako ipak planiram lizirati stanice.

Za pročišćavanje proteina važna je sterilna tehnika ako namjeravate uzorak čuvati neko vrijeme. Proteaze u okolišu i na vašem uzorku/sebi mogu nakratko uništiti sav vaš naporan rad.


Nikada ne vjerujte u potpunosti svojim antibioticima. Kao prvo, oni često neće zaustaviti gljivice, što će uništiti vaše uzorke čak i u prisutnosti nekih prilično gadnih kemikalija. Ako koristite neki od luđih antibiotika (ampicilin je najbolji primjer), antibiotik se s vremenom razgrađuje. Posebno su problem B-laktami jer se B-laktamaza izlučuje u medije. Do drugog dana kulture, u osnovi neće ostati ampicilin!


Za većinu pripravaka plazmida nije potrebna stroga sterilna tehnika. Antibiotici u medijima odabiru bakterije koje nose rezistentni plazmid, tako da obično ne morate brinuti o ulasku drugih organizama.

Međutim, kada koristite nekoliko vrsta plazmida, morate biti oprezni kako biste izbjegli unakrsnu kontaminaciju, jer je svaki plazmid otporan, pa će bakterije transformirane s plazmidom A sasvim dobro živjeti na ploči plazmida B. Kada oblažete svoje stanice, prođite kroz stanicu kroz plamen. Ne mora biti službeni Bunsenov plamenik, sasvim sam dobro koristio alkoholni plamenik.

Ako napravite veliku bocu medija i koristite male alikvote odjednom, tada se boca treba otvoriti samo unutar haube.


BacMam sustav za visoku razinu ekspresije rekombinantnih topljivih i membranskih glikoproteina za strukturna istraživanja

Bakulovirus posredovana genska transdukcija stanica sisavaca (BacMam) nova je tehnika za brzu ekspresiju rekombinantnog proteina u stanicama sisavaca. Konstruirali smo dva vektora za prijenos bakulovirusa koji uključuju nekoliko regulatornih elemenata za transkripciju sisavaca neophodnih za ekspresiju proteina na visokim razinama u stanicama sisavaca. Pomoću ovih vektora pokazujemo da se sustav BacMam u kombinaciji s 293 GnTI -stanične linije može koristiti za proizvodnju miligramskih količina topljivih glikoproteina. Štoviše, za pokuse kristalizacije, pročišćeni glikoproteini osjetljivi su na EndoH tretman što rezultira gubitkom najvećeg dijela priloženog N-povezana glikozilacija. Osim toga, također smo pokazali da se kombinacija BacMam sustava i 293 GnTI -stanične linije može koristiti za proizvodnju miligramskih količina GPCR -proteinskog ligandnog kompleksa pogodnog za pokuse kristalizacije.


Postavljanje pozornice - povijesna i evolucijska perspektiva

Koliko smo odstupili od metode kloniranja prirode? Čak i izostavljanje većine drugih oblika biljnog i životinjskog svijeta i usredotočenje na kralježnjake - životinje s kralježnicom - primjeri klonova obiluju prirodom. Jednojajčani blizanci su očiti primjeri, ali možda su intrigantniji armadili, u kojima su potomci u leglu svi klonovi izvedeni iz jedne zigote (9). Najjednostavniji oblik umjetnog kloniranja je cijepanje embrija - odvajanje blastomera ranog embrija i stvaranje dva ili više manjih embrija. Početne studije provedene su kako bi se postavila ključna pitanja u vezi s kontrolom razvoja loze: Kada je određena sudbina stanice i koliko je ta sudbina plastična? Studije na vodozemcima, zečevima i miševima sugerirale su da su vrlo rane faze cijepanja (od dvije do četiri stanice) bile fleksibilne i da bi svaki blastomer mogao dati održivu blastocistu. U kasnijim fazama, blastomeri više nisu mogli neovisno stvarati održivu blastocistu zbog gubitka mase jer je svaki blastomer prošao cijepanje. To ne znači da jezgra blastomera nije bila u stanju usmjeriti potpuni razvoj, već prije nije mogla zaustaviti razvojni sat i zamijeniti izgubljenu masu prije nastavka. Prije stupnja blastociste, stanice u ranom embriju, zvane blastomere, dijele se bez povećanja mase između svake diobe: dakle pojam cijepanja - svaka se stanica cijepa na pola. Ovo ograničenje dovelo je do očitog pitanja: Ako osigurate dodatnu masu, mogu li kasnije postavljeni blastomeri - ili prikladnije - mogu li jezgre kasnije etapnog blastomera usmjeriti potpuni razvoj do stupnja blastociste i biti sposobni za nastavak razvoja što rezultira normalnim, živim potomstvo? Pionirska istraživanja 1950 -ih i 1960 -ih na žabama pokazala su da jezgre od embrija do stupca punoglavca mogu usmjeriti normalan razvoj, rezultirajući odraslim pojedincima, ali da su jezgre iz tkiva odraslih sposobne usmjeriti razvoj samo do stupca punoglavca (10, 11). Unatoč neuspjehu dobivanja odraslih osoba nakon nuklearnog prijenosa odraslih stanica, studije su pokazale razvojnu plastičnost diferenciranih, somatskih staničnih jezgri. Nepoznati (u to vrijeme) regulatorni mehanizmi koji kontroliraju ekspresiju gena specifičnih za stanice mogli bi se vratiti natrag u stadij embrija.

Sustavna ispitivanja embrionalne plastičnosti sisavaca nisu se mogla provoditi sve dok 1960 -ih i 1970 -ih nisu uspostavljeni odgovarajući uvjeti kulture in vitro (12 ⇓ –14). Nakon toga, kontroverzne studije 1970 -ih sugerirale su da jezgre iz stanica koje su prošle prvu diferencijaciju loze (to jest, stanice koje su formirale unutarnju staničnu masu) mogu usmjeriti normalan razvoj ako se zamijene sa zigotičkom jezgrom (15). Međutim, neuspjeh drugih istraživačkih skupina da ponove ove studije doveo je neke znanstvenike do zaključka da jezgre sisavaca nakon aktivacije embrionalnog gena nisu bile u mogućnosti usmjeriti razvoj zbog nepovratnih promjena u programiranju (16). U ovom trenutku, napredak reproduktivnih tehnologija koji uključuje domaće životinje, prvenstveno ovce i goveda, omogućio je znanstvenicima u životinjama da, uz postavljanje pitanja o razvojnoj plastičnosti, prilagode takve tehnike kao što su cijepanje embrija i kloniranje blastomera, s naglaskom na poboljšanju učinkovitosti proizvodnje i genetskom napretku. Budući da razvojni biolozi, usredotočeni na tradicionalnije modele (npr. Miš i žabe), ne čitaju više znanstvene časopise vezane za poljoprivredu, napredak koji su postigli znanstvenici na životinjama bio je uglavnom neprepoznat sve dok nije objavljena Dollyjeva zaslužna publikacija u Priroda (6). Iako je činjenica da bi jezgra odrasle osobe mogla doista usmjeriti normalan razvoj (rezultirajući živim potomstvom) bila revolucionarna za razvojnu biologiju, slijedila je niz otkrića koja su sugerirala takvu mogućnost (slika 2).

Vremenski okvir ključnih točaka tijekom razvoja SCNT -a u domaćem stočarstvu (6, 17 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ 33 33 –33).

Prvi pokušaji umjetnog kloniranja domaćih životinja uključivali su cijepanje embrija. Steen Willadsen pokazao je da se blizanci mogu proizvesti u ovaca (17) i goveda (18) nakon cijepanja embrija u fazi cijepanja i prijenosa demi embrija u primatelje. Ove su studije pokazale da se mogu dobiti trojke, pa čak i četvorke, iako na nižim frekvencijama zbog gubitka stanične mase. Kako bi prevladao ovo ograničenje, Willadsen je upotrijebio izmijenjenu verziju tehnike prijenosa nuklearnog oružja koja je korištena u prethodnim studijama kloniranja vodozemaca (Slika 1). Ukratko, DNK oocita je uklonjena aspiracijom dijela ooplazme koji sadrži kromosome, čime je formiran citoplast, a donorska stanica je postavljena uz ooplazmatsku membranu, a citoplast i donorska stanica spojene su zajedno. Ovim postupkom Willadsen je nakon prijenosa rekonstruiranih embrija u surogate dobio živu janje (19) i telad (20). Nakon toga, nekoliko drugih istraživačkih skupina povezanih s poljoprivrednom industrijom počelo je istraživati ​​mogućnosti prijenosa nuklearnih embrija i embrionalnih stanica, postižući uspjeh s postupno kasnijim fazama embrija (Tablica 2). Godine 1996. istraživači s Instituta za istraživanje Roslin izvijestili su o uspješnoj proizvodnji žive janjadi korištenjem dugo uzgojenih embrionalnih (21), pa čak i transgenih (22) stanica. Ubrzo je nakon ovih postignuća došlo izvješće o proizvodnji janjetine (Dolly) pomoću uzgojenih somatskih stanica dobivenih od odrasle osobe (6). Iako je mnogo učinjeno na niskoj učinkovitosti prijenosa nuklearnih somatskih stanica (SCNT) - Doly je bio jedini živi potomak koji je rezultat 29 prenesenih rekonstruiranih embrija za koje je manipulirano 247 oocita - činjenica da je proizvedeno živo janje još uvijek je zapanjujuća .

Upotreba progresivno sve većeg broja napredovanih jezgri za SCNT u goveda

Da bi se u potpunosti shvatile prepreke umjetnom kloniranju, najprije je potrebno razumjeti procese gametogeneze i oplodnje. Tijekom razvoja sisavaca, primordijalne zametne stanice u fetusu u razvoju migriraju u gonadalne grebene i, ovisno o spolu fetusa, tvore oogoniju ili spermatogoniju. Uzorci metilacije DNA uspostavljeni su tako da su spermiji hipermetilirani, a oociti hipometilirani u usporedbi sa somatskim stanicama (34). Nakon oplodnje, DNK sperme podliježe aktivnoj demetilaciji, a majčina DNA pasivnoj demetilaciji. Neki geni zadržavaju očev ili majčinski otisak tako da se aktivna transkripcija događa samo s jednog roditeljskog kromosoma. Ti utisnuti geni često igraju kritičnu ulogu u placentaciji: npr. Utisnuti faktor rasta, IGF2, izražava očinski, a ne majčinski alel, dok se receptor , IGF2R, majčinski je izražen (35). Iako se ti otisci općenito opisuju njihovim uzorcima metilacije DNA, složeni mehanizmi i razine utiskivanja, uključujući modifikacije histona, još uvijek se dešifriraju. Zašto je otisak važan tijekom umjetnog kloniranja? Uostalom, donorske stanice imaju komplete kromosoma izvedene i od majke i od oca. Tijekom razvoja, kako se stanice podvrgavaju diferencijaciji loze, neprestano se uspostavljaju epigenetske promjene koje mijenjaju genetski program za ekspresiju gena specifičnu za ćeliju. Ove promjene uključuju više razina epigenetskih promjena, uključujući metilaciju DNA i posttranslacijske modifikacije histonskih proteina (slika 3). Tijekom reprogramiranja ooplazmom ti se uzorci moraju vratiti na zigotičke obrasce. Ovaj zahtjev opterećuje sposobnosti faktora reprogramiranja unutar ooplazme kako su se razvili kako bi resetirali očeve i majčinske gametske epigenetske obrasce, a ne one somatske stanice. Stoga ne čudi da oociti nisu u stanju u potpunosti i ispravno reprogramirati somatski epigenom. Zapravo je iznenađujuće što su sposobni postići dovoljno odgovarajući epigenom koji omogućuje potpuni razvoj. Kako su primijetili drugi autori, priroda dopušta određenu fleksibilnost u epigenomu i ekspresiji gena tijekom rasta i razvoja (28). Kao što se moglo očekivati, pronađeno je da klonovi koji zataje tijekom trudnoće i/ili imaju fiziološke abnormalnosti imaju abnormalne epigenetske obrasce, dok oni koji uspijevaju imaju relativno normalan obrazac (28, 36). Zanimljivo je i napomenuti da su gestacijski gubici i abnormalnosti uočeni u SCNT -u također zabilježeni tijekom razvoja in vitro proizvodnje i tehnike zametaka u domaćim vrstama 1990 -ih. U ovaca i goveda, zahvaćeni potomci bili su tipično veći od normalnih, pa je skovan izraz "sindrom velikog potomstva" (37). Utvrđeno je da je izloženost serumu i kokulturi promijenila obrasce epigenetske metilacije embrija (38, 39). S poboljšanjima u medijima za uzgoj, čini se da učestalost velikog potomstva nakon proizvodnje embrija in vitro više nije pitanje koje je nekad bilo, iako su suptilnije epigenetske promjene koje mogu imati dugoročne posljedice na zdravlje potomaka od velikog interesa. Brojne istraživačke skupine istražuju ove suptilne epigenetske promjene koje se mogu dogoditi tijekom gameta, embrija i rane trudnoće s potencijalno dugoročnim posljedicama na potomstvo (40, 41).

Epigenetski čimbenici reguliraju dostupnost DNA transkripcijskim strojevima (transkripcijski faktori, polimeraze itd.) I uključeni su u kontrolu ekspresije gena specifičnog za stanično tkivo.


REZULTATI

AgNAT6 je kloniran iz zbirke cDNA srednjih crijeva ličinki komaraca 4. stadija (GenBank CDS AJ626713 i PID CAF25029). Ima ORF iz 1953 bp koji kodira protein dug 650 aminokiselina s procijenjenom molekulskom masom od 71,5 kDa i pI od 7,31. Najbliži je filogenetski u odnosu na ranije okarakterizirani Na + –fenilalanin, fenil razgranati supstrat supstrata AgNAT8 (Slika 1, sličnost po parametrima 52,9%, identična mjesta 343, 51,7%) i novokarakterizirani član potporodice NATs iz obitelji SLC6. Geni AgNAT6 i AgNAT8 proizlaze iz najnovijeg dupliciranja u grupi AgNAT-SLC6 (slika 2). Oni su prvi uzvodni i posljednji nizvodni geni NAT klastera sa sedam gena koji je mapiran na pozitivnom lancu 3L kromosoma između 12,09 i 12,16 Mb položaja (Meleshkevitch i sur., 2006), na temelju genoma bilješka (Holt i sur., 2002.).

Usklađivanje AgNAT6 s odabranim transporterima aminokiselina insekata i bakterija (NAT). Transmembranske domene (TMD1–12) i očuvane strukturne značajke, uključujući mjesta interakcije supstrata, identificirane su nakon usklađivanja AgNAT6 s odabranim insektima i prokariotskim NAT -ovima, uključujući LeuTAa slijed (ova slika), koji je podržan strukturnim poravnanjem, i pristajanje podloge (nije prikazano). Poravnanje je generirano softverom Geneious Pro 4.5 (Biomatters, Auckland, Novi Zeland) s manjim ručnim poboljšanjem. Povećanje intenziteta pozadine ukazuje na povećanje sličnosti sekvenci. Zeleni trokuti označavaju mjesta vezivanja supstrata, crveni i smeđi rombi su prvo i drugo mjesto interakcije Na +, ljubičasti kvadrati predstavljaju mjesta koja vežu Cl. Tamno crvene strelice su transmembranske spirale. Žute trake su sub-membranske (EL, izvanstanična petljaIL, unutarstanična petlja) spirale.

Usklađivanje AgNAT6 s odabranim transporterima aminokiselina insekata i bakterija (NAT). Transmembranske domene (TMD1–12) i očuvane strukturne značajke, uključujući mjesta interakcije supstrata, identificirane su nakon usklađivanja AgNAT6 s odabranim insektima i prokariotskim NAT -ovima, uključujući LeuTAa slijed (ova slika), koji je podržan strukturnim poravnanjem, i pristajanje podloge (nije prikazano). Poravnanje je generirano softverom Geneious Pro 4.5 (Biomatters, Auckland, Novi Zeland) s manjim ručnim poboljšanjem. Povećanje intenziteta pozadine ukazuje na povećanje sličnosti sekvenci. Zeleni trokuti označavaju mjesta vezivanja supstrata, crveni i smeđi rombi su prvo i drugo mjesto interakcije Na +, ljubičasti kvadrati predstavljaju mjesta koja vežu Cl. Tamno crvene strelice su transmembranske spirale. Žute trake su sub-membranske (EL, izvanstanična petljaIL, unutarstanična petlja) spirale.

AgNAT6 ima tipičnu strukturu SLC6 s 12 transmembranskih domena i unutarstaničnim krajevima C i N (slika 1). Dvanaest koordiniranih ostataka organskog supstrata može se identificirati na temelju poravnanja sekvencijalne strukture AgNAT6, AeAAT1 (Boudko i sur., 2005.a), AgNAT8 (Meleshkevitch i sur., 2006.) i TnaT (Androutsellis-Theotokis i sur., 2003.) s kristaliziranim LeuTAa(Yamashita i sur., 2005.) (slika 1 Tablica 1). Ostaci koji međusobno djeluju sa supstratom sadrže iznenađujuće očuvan uzorak, s devet apsolutno očuvanih mjesta između AgNAT6 i AgNAT8 (Tablica 1). Tri različite aminokiseline u transmembranskim domenama 1, 6 i 8 mogle bi biti odgovorne za triptofansku selektivnost AgNAT6. Konkretno, oni uključuju: L91, jer se ovo mjesto razlikuje od odgovarajućeg mjesta M95 u AgNAT8 T333, koje se razlikuje od AgNAT8 S339 i identično je s TnaT T234, te G437 koje je jedinstveno za AgNAT6 (d, i i u označavaju odgovarajuća mjesta u tablici 1). Ova tri položaja odgovaraju N21, S356 i A358 LeuT -aAa. Sve tri zamjene pomažu u smanjenju volumena bočnog lanca i odgovarajućem povećanju volumena ovojnice koja veže supstrat, što je u korelaciji sa sposobnošću prihvaćanja indola veće veličine vs supstrati razgranati fenolom (slika 3A, umetnuto).

Poravnavanje ovojnica koje vežu supstrat odabranih prokariotskih i eukariotskih NAT-ova

TMD br. . 11111-333-666666-888 . Fenotip.
AgNAT6 LS-LG-V-Y-FF-T-F-S-G Trp specifičan
AgNAT8 MS-LG-L-Y-FF-S-F-S-A Specifičan
AeAAT1 MS-LG-V-Y-FS-S-M-S-A Phe široki
TnaT AA-LG-V-Y-FF-T-V-S-S Trp specifičan
LeuT NA-LG-V-Y-FF-S-F-S-A Leu široka
Stranice: dc-cc-c-c-cc-i-c-c-u
TMD br. . 11111-333-666666-888 . Fenotip.
AgNAT6 LS-LG-V-Y-FF-T-F-S-G Trp specifičan
AgNAT8 MS-LG-L-Y-FF-S-F-S-A Specifičan
AeAAT1 MS-LG-V-Y-FS-S-M-S-A Phe široki
TnaT AA-LG-V-Y-FF-T-V-S-S Trp specifičan
LeuT NA-LG-V-Y-FF-S-F-S-A Leu široka
Stranice: dc-cc-c-c-cc-i-c-c-u

NAT, transporter hranjivih aminokiselina Ae, Aedes aegypti Ag, Anopheles gambiae TMD, transmembranska domena

Mjesta interakcije sa supstratom: c, očuvana u AgNAT6 i AgNAT8 d, različita u AgNAT6 i AgNAT8 i, identična u Trp-specifičnim eukariotskim i prokariotskim transporterima u, jedinstvena u AgNAT6

Ekspresija svih sedam AgNAT -ova, devet AeNAT -ova i šest DmNAT -a potvrđena je molekularnim kloniranjem iz specifičnih zbirki cDNA (svih 22 sekvenci klonova dostupno je u bazi podataka NCBI, vidi sliku 1 za pristupne brojeve NCBI -a), ali trenutno su samo dva AgNAT -a bila karakteriziran heterolognom ekspresijom ranije, AgNAT8 (Meleshkevitch i sur., 2006.), a sada, AgNAT6. Primjena aromatičnih aminokiselina proizvela je jedva zamjetljivu struju u naivnim i DW injektiranim X. laevis oocita (N& gt100 pri –30 mV Hp) i posredovale su velike struje izazvane aromatskim aminokiselinama od 20–150 nA (slika 3A). Za razliku od AgNAT8 koji preferira fenilalanin, AgNAT6 je reagirao sa znatno većim strujama nakon primjene triptofana i 5-HTP (slika 3B), što ukazuje na veću transportnu brzinu za podloge razgranate s indolom nego na fenolu. Među metabolitima izvedenim iz Phe samo je Trp (dodavanje hidroksilne skupine na kraj 6-ugljikovog aromatskog prstena s odgovarajućim povećanjem molekulskog volumena) proizveo struje koje su slične strujama induciranim supstratom iz grane indola (slika 3A). Zamjenom Na + s Li + ili K + uklonjene su triptofanom inducirane struje pri –30 mV Hp (slika 3D). Međutim, produžena struja -napon (I – V) grafikon je otkrio da AgNAT6 može generirati velike struje K + povezane sa supstratom pri transmembranskim naponima negativnijim od –40 mV (slika 3E). Pokusi preuzimanja izotopa potvrdili su preuzimanje AgNAT6 organskog supstrata sa značajno višim omjerom unosa za triptofan obilježen izotopom nego za fenilalanin (slika 3F).

Filogenetski položaj AgNAT6 u stablu obitelji nosača otopljene tvari 6 (SLC6). Drvo uključuje 98 članova SLC6 iz sedam kompletiranih genoma, uključujući jednog sisavca, tri insekta dipterana, jednu nematodu i dva prokariotska genoma, dodane su i dvije karakterizirane lepidopteranske sekvence NAT. Evolucijska povijest zaključena je pomoću UPGMA metode (Sneath i Sokal, 1973). Prikazano je optimalno stablo sa zbrojem duljine grana = 63,23. Postotak repliciranih stabala u kojima su povezani taksoni grupirani zajedno u bootstrap testu (2000 ponavljanja) prikazan je uz grane (Felsenstein, 1985). Stablo je nacrtano u mjerilu, s duljinama grana u istim jedinicama kao i evolucijske udaljenosti korištene za zaključivanje filogenetskog stabla. Evolucijske udaljenosti izračunate su Poissonovom korekcijskom metodom (Zuckerkandl i Pauling, 1965.) i u jedinicama su broja zamjena aminokiselina po mjestu. Varijacija brzine među mjestima modelirana je gama distribucijom (parametar oblika = 1). Sve pozicije koje sadrže praznine poravnanja i nedostajuće podatke eliminirane su samo usporedbom u paru (opcija brisanja u paru). U konačnom skupu podataka bilo je ukupno 566 pozicija. Filogenetske analize provedene su u MEGA4 (Tamura i sur., 2007.). Linije prikazuju pristupne brojeve NCBI -a praćene proizvoljnim definicijama očitih ortologa i kloniranih transportera (prikazano podebljano). Kratice: Ae, Aedes aegypti Ag, Anopheles gambiae Ce, Caenorhabditis elegancija Dm, Drosophila melanogaster Mm, Mus musculus Gđa, Manduca sexta NTT -i, transporteri neurotransmitera. Raspravljani prijevoznici su podcrtani. Beskralježnjački NAT -i prikazani su različitim bojama fontova.

Filogenetski položaj AgNAT6 u stablu obitelji nosača otopljene tvari 6 (SLC6). Stablo uključuje 98 članova SLC6 iz sedam kompletiranih genoma, uključujući jednog sisavca, tri insekta dipterana, jednu nematodu i dva prokariotska genoma, dodane su i dvije karakterizirane lepidopteranske sekvence NAT. Evolucijska povijest zaključena je pomoću UPGMA metode (Sneath i Sokal, 1973). Prikazano je optimalno stablo sa sumom duljine grane = 63,23. Postotak repliciranih stabala u kojima su povezani taksoni grupirani zajedno u bootstrap testu (2000 ponavljanja) prikazan je uz grane (Felsenstein, 1985). Stablo je nacrtano u mjerilu, s duljinama grana u istim jedinicama kao i evolucijske udaljenosti korištene za zaključivanje filogenetskog stabla. Evolucijske udaljenosti izračunate su Poissonovom korekcijskom metodom (Zuckerkandl i Pauling, 1965.) i u jedinicama su broja zamjena aminokiselina po mjestu. Varijacija brzine među mjestima modelirana je gama distribucijom (parametar oblika = 1). Sve pozicije koje sadrže praznine poravnanja i nedostajuće podatke eliminirane su samo usporedbom u paru (opcija brisanja u paru). U konačnom skupu podataka bilo je ukupno 566 pozicija. Filogenetske analize provedene su u MEGA4 (Tamura i sur., 2007.). Linije prikazuju pristupne brojeve NCBI -a praćene proizvoljnim definicijama očitih ortologa i kloniranih transportera (prikazano podebljano). Kratice: Ae, Aedes aegypti Ag, Anopheles gambiae Ce, Caenorhabditis elegancija Dm, Drosophila melanogaster Mm, Mus musculus Gđa, Manduca sexta NTT -i, transporteri neurotransmitera. Raspravljani prijevoznici su podcrtani. Beskralježnjački NAT -i prikazani su različitim bojama fontova.

Elektrokemijska svojstva mehanizma AgNAT6 izražena u Xenopus laevis jajne stanice. (A) Primjer strujanja induciranih supstratom dobivenih iz reprezentativnog oocita. Svi supstrati su superkondenzirani pri koncentracijama 1 mmol l –1 u 98 mmol l – 1 mediju NaCl na –30 mV koji ima transmembranski naponski potencijal. (B) Normalizirane struje inducirane supstratom (šipke su srednja vrijednost ± s. D. Za N& gt3 pokusi i oociti). (C) Neobični pozitivni odgovori, koji su se često, ali ne uvijek, primijetili nakon primjene l -Phe i l -DOPA 6-8 dana nakon injekcije cRNA. (D) Ionska ovisnost Trp-inducirane struje. Milimolarne koncentracije glavne komponente soli u perfuzijskim otopinama prikazane su gore navedenim redoslijedom trenutnih tragova. (E)I – V grafikoni interakcija AgNAT6 s Trp-om pri specifičnim sastavima anorganskih iona grafikoni predstavljaju odnose struja-napon nakon oduzimanja Trp-neovisne komponente struje. I – V plohe koje se odnose na perfuzijske otopine koje sadrže različite glavne soli prikazane su različitim stilovima linija (vidi ključ). (F) Relativni omjeri unosa izračunati nakon 10-minutne izloženosti oocita ubrizganih AgNAT6 (ispunjene šipke) i oocita ubrizganih destiliranom vodom (DW) prema specificiranoj podlozi označenoj izotopom (stupci su normalizirani prosječni omjer unosa ± sd, N=3).

Elektrokemijska svojstva mehanizma AgNAT6 izražena u Xenopus laevis jajne stanice. (A) Primjer strujanja induciranih supstratom dobivenih iz reprezentativnog oocita. Svi supstrati su superkondenzirani pri koncentracijama 1 mmol l –1 u 98 mmol l – 1 mediju NaCl pri –30 mV koji ima transmembranski naponski potencijal. (B) Normalizirane struje inducirane supstratom (šipke su srednja vrijednost ± s. D. Za N& gt3 pokusi i oociti). (C) Neobični pozitivni odgovori, koji su se često, ali ne uvijek, primijetili nakon primjene l -Phe i l -DOPA 6-8 dana nakon injekcije cRNA. (D) Ionska ovisnost Trp-inducirane struje. Milimolarne koncentracije glavne komponente soli u perfuzijskim otopinama prikazane su gore navedenim redoslijedom trenutnih tragova. (E)I – V grafikoni interakcija AgNAT6 s Trp-om pri specifičnim sastavima anorganskih iona grafikoni predstavljaju odnose struja-napon nakon oduzimanja Trp-neovisne komponente struje. I – V plohe koje se odnose na perfuzijske otopine koje sadrže različite glavne soli prikazane su različitim stilovima linija (vidi ključ). (F) Relativni omjeri unosa izračunati nakon 10-minutne izloženosti oocita ubrizganih AgNAT6 (napunjene šipke) i destilirane vode (DW) unesene (specificirane podloge označene izotopom (stupci su normalizirani prosječni omjer unosa ± sd, N=3).

Mehanizam AgNAT6 ima zasićenu kinetiku (slika 4A). Triptofan ima izrazito veći prividni afinitet od svih ostalih ispitanih podloga (K50 Trp = 1,3 μmol l –1 u usporedbi s K0.5 5-HTP = 270 & ltK0.5 Tyr & ltK0.5 Phe & ltK0.5 DOPA & ltK0.5 Leu = 890μmol l –1). Redovi reakcije translokacije organskog supstrata (Hillova konstanta η) određeni pri 98 mmol l –1 Na + koncentracije su svi slični i približavaju se 1 (slika 4B).

AgNAT6 testiran na dvije različite koncentracije triptofana pokazao je mali porast K50 Na+ na 3 mmol l –1 Trp vs 0,3 mmol l –1 Trp (slika 4C). Neočekivano smo identificirali sposobnost AgNAT6 da ispolji Hill koeficijente od 2 i 1 Na + za translokacije triptofana pri koncentracijama od 0,3 odnosno 3 mmol l – 1 (slika 4C). Ovo "klizanje" može ukazivati ​​na promjenu željene transportne stehiometrije u mehanizmu AgNAT6: 1 Na +: 1 aminokiselina pri visokim koncentracijama organskih supstrata i 2 Na +: 1 aminokiselina pri niskim koncentracijama organskih supstrata. AgNAT6 je pokazao nelinearnu ovisnost o pH s relativno visokim prijenosom pri umjereno kiseloj (pH 6) i jako alkalnoj (pH 8–9) vs neutralne (pH 7) vanjske otopine.

Kinetička svojstva i pH ovisnost AgNAT6. Struje posredovane AgNAT6 u funkciji koncentracije odabranih organskih supstrata (A) i natrijevih iona pri koncentracijama triptofana (C) od 0,3 i 3 mmol l – 1. Krivulje su nelinearna regresija podataka izračunatih iz Hillove jednadžbe: f = sjekira η /(K0.5 η +x η) 3 mmol l –1 Trp (ispunjeni krugovi), η = 0,98 ± 0,14, K0.5= 25,27 ± 6,38 i 0,3 mmol l –1 Trp (otvoreni krugovi), η = 1,90 ± 0,21, K0.5= 38,46 ± 3,47 u C. (B) Procijenjene konstante afiniteta (stupci su srednja vrijednost ± s.d., N& gt3) i Hillove konstante (krugovi su srednja vrijednost ± s.d., N& gt3) za odabrane organske podloge. (D) pH ovisnost AgNAT6 (krugovi su srednja vrijednost ± s.d., N& gt3).

Kinetička svojstva i pH ovisnost AgNAT6. Struje posredovane AgNAT6 u funkciji koncentracije odabranih organskih supstrata (A) i natrijevih iona pri koncentracijama triptofana (C) od 0,3 i 3 mmol l – 1. Krivulje su nelinearna regresija podataka izračunatih iz Hillove jednadžbe: f = sjekira η /(K0.5 η +x η) 3 mmol l –1 Trp (ispunjeni krugovi), η = 0,98 ± 0,14, K0.5= 25,27 ± 6,38 i 0,3 mmol l –1 Trp (otvoreni krugovi), η = 1,90 ± 0,21, K0.5= 38,46 ± 3,47 u C. (B) Procijenjene konstante afiniteta (stupci su srednja vrijednost ± s.d., N& gt3) i Hillove konstante (krugovi su srednja vrijednost ± s.d., N& gt3) za odabrane organske podloge. (D) pH ovisnost AgNAT6 (krugovi su srednja vrijednost ± s.d., N& gt3).

In situ hibridizacija probavnog kanala larvi s antisens sondom AgNAT6 otkrila je relativno visoku akumulaciju transkripta AgNAT6 u srcu, želučanoj slijepoj crijevi i stražnjem srednjem crijevu (slika 5A). Slabije, ali uočljivo označavanje bilo je prisutno u prednjoj sredini crijeva (slika 5A). Jaka in situ signali hibridizacije detektirani su u neuronskom pleksusu glave larve povezanom s kemo-, vizualnim i mehano-senzornim modalitetima te u neuropili ventralne živčane vrpce (slika 5B, C). Snažan signal otkriven je u nekoliko pojedinačnih neurona nakon snimanja pripravaka za cijelu montažu niske rezolucije (slike nisu prikazane). Vrlo snažan signal otkriven je i u probavnom kanalu larve 2. i 3. stupnja, osobito kada su mlade ličinke bile izložene ograničenim hranjivim uvjetima (slika 5D). qPCR je potvrdio sveprisutnu ekspresiju AgNAT6 s vrlo jakim varijacijama transkripcijskih veličina AgNAT6 specifičnih za tkivo i razvojno stadij (sl. 5E).


Materijali i metode

Životinje

Ženke miševa B6D2F1 (C57BL/6 × DBA/2 hibrid) kupljene su od Japana SLC (Shizuoka, Japan) i korištene kao davatelji oocita, kumulusnih stanica i perifernih krvnih stanica. Za prikupljanje perifernih krvnih stanica, transgenih ženki miševa sa okto4-zelenim fluorescentnim proteinom (GFP) (GOF18-ΔPE-GFP [10]), transgenih ženki miševa Dppa3-Venus, ženkom miša 129XB6-P i ženkom miša 129XB6-F rabljeno. Soj Oct4-GFP izveden je iz križanja izvornih transgenih mužjaka sa ženkama B6D2F1 za> gt10 generacija u našem laboratoriju i korišten je u dobi od 8-45 tjedana. Soj Dppa3-Venera nastao je ubrizgavanjem pronuklearne DNA u embrije B6D2F1 × B6 u našem laboratoriju. Mužjak osnivača miša bio je paren sa ženkama B6, a transgene ženke na F2 i F.3 generacije korištene su kao donatori u dobi od 50 do 80 tjedana. Sojevi 129XB6-P i 129XB6-F bili su rekombinantni inbred sojevi generirani iz hibrida između 129 i C57BL/6 sojeva, a ženke miševa u dobi od približno 8 tjedana nabavljene su iz zaliha u centru za biološke resurse RIKEN. ICR ženke miševa (CLEA Japan, Tokio, Japan), stare 8–16 tjedana, korištene su kao primatelji prijenosa embrija. Životinje su bile smještene u kontroliranim svjetlosnim uvjetima (0700–2100 h) i održavane su u uvjetima bez specifičnih patogena. Sve pokuse na životinjama odobrilo je Povjerenstvo za eksperimente na životinjama pri Institutu RIKEN Tsukuba i provedeni su u skladu s načelima vodstva odbora.

Priprema ćelija donatora

Peripheral blood was collected from the tail vein using a heparinized calibrated pipette (45 μl Drummond Scientific Company, Broomall, PA). Blood was transferred to a 1.5-ml tube containing 10–20 μl of 0.1 M EDTA. Collected peripheral blood was treated with erythrocyte-lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 2 mM EDTA, pH 7.2) and washed three times by centrifugation (1200 × g for 5 min). After centrifugation, the pellet containing leukocytes and erythrocyte debris was resuspended in Hepes-buffered potassium simplex-optimized medium (KSOM) [ 11]. Then, normal-looking leukocytes in the cell suspension were used as nuclear donor cells. To distinguish between lymphocytes and other cells (mostly granulocytes), the cell suspension was treated with lymphocyte-specific fluorescein isothiocyanate (FITC)-conjugated, pure Phytolacca americana-derived lectin (PWM EY Laboratories, Inc., San Mateo, CA) and was examined using a fluorescence microscope or by fluorescence-activated cell sorting (FACS) analysis. We also tried to separate specific leukocyte types by their sizes. To determine the accuracy of such size-dependent separation, cells selected using an inverted microscope were placed on a glass slide and compressed on the surface using a CytoSpin III cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA) at 600 rpm for 2 min. The cells were stained with May-Grünwald Giemsa stain to identify leukocyte types.

Nuclear Transfer

Nuclear transfer was carried out as described previously [ 12, 13]. Briefly, B6D2F1 females were superovulated by injection of 7.5 IU of equine chorionic gonadotropin and 7.5 IU of human chorionic gonadotropin (hCG) at a 46- to 52-h interval. At 14–17 h after receiving the hCG injection, the mice were euthanized, cumulus-oocyte complexes were collected from the oviducts, and cumulus cells were removed by suspending them in KSOM containing 0.1% bovine testicular hyaluronidase. Oocytes were enucleated in Hepes-buffered KSOM containing 7.5 μg/ml cytochalasin B. The donor nuclei were injected into enucleated oocytes by using a piezo-driven micropipette (Prime Tech Ltd., Ibaraki, Japan). After culture in KSOM for 1 h, injected oocytes were incubated in Ca 2+ -free KSOM containing 2.5 mM SrCl2, 5 μg/ml cytochalasin B, and 50 nM trichostatin A (TSA Sigma-Aldrich, St. Louis, MO) under 5% CO2 in air at 37°C for 1 h. Next, the oocytes were incubated in KSOM containing 5 μg/ml cytochalasin B and 50 nM TSA for 5 h, followed by incubation in KSOM containing 50 nM TSA alone for 2 h. After being washed, the oocytes were cultured in KSOM under 5% CO2 in air at 37°C. Cloned embryos that had reached the 2-cell or 4-cell stage after 24 h or 48 h in culture, respectively, were transferred into the oviducts of pseudopregnant ICR female mice at 0.5 day postcopulation (dpc the day following sterile mating with a vasectomized male mouse). The pregnant females were euthanized on 19.5 days postcopulation and examined for fetuses and placentas in their uteri. In the last experiment using a Dppa3-Venus female mouse, we used a gene knockout strategy with short interfering RNA (siRNA) against Xist. Reconstructed oocytes at approximately 6–7 h after activation were injected with siRNA (25 μM) against Xist using a piezo-driven micropipette, as described previously [ 14].

Fluorescence-Activated Cell Sorting

After red blood cells were lysed, peripheral blood cells were stained with FITC-conjugated PWM lectin and phycoerythrin-conjugated anti-Mac-1, anti-Gr-1, anti-B220, anti-CD4, or anti-CD8 antibody (eBioscience, San Diego, CA). The stained cells were analyzed and sorted using a FACSAria instrument (BD Biosciences, San Jose, CA).

Analysis of Rearranged Immunoglobulin Variable Region Genes

Recombination of the immunoglobulin heavy chain region was detected as described by Rohatgi et al. [ 15] with slight modifications as follows. The first PCR amplification was carried out using a Multiplex PCR kit (TaKaRa Bio Inc., Kyoto, Japan) with a mixture of the “external” VH primers (either of two sets for VH1–VH7 and VH8–VH16) and the “external” JH primer as described [ 15]. To obtain clear results, we next performed nested PCR with a mixture of the “internal” VH primers and the “internal” JH primers under the same PCR conditions as described for the first PCR. The PCR products were analyzed using 2% agarose gel electrophoresis.

Statistička analiza

Developmental rates of embryos were analyzed using the chi-square test with Yates correction. The size of each type of leukocyte was analyzed by one-way analysis of variance, followed by the Tukey-Kramer procedure for multiple comparisons. A P value of <0.05 was considered statistically significant.


Zaključci

The present results show the subtraction step performed to obtain the SSH library used was appropriate, with a significant reduction achieved in the number of constitutive or downregulated sequences. A significant proportion of the amplified DNA sequences generated by the PK-profiling primers were similar to PK genes of related species. They were specifically expressed in the MN841801–1 line after their induction with P. coronata. PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata and perhaps by other pathogens.


Sperm superiority of reprogramming

Sperm are highly differentiated, transcriptionally inert cells with minimal cytoplasm, and they contain a suite of unique RNAs that are delivered to oocytes. These RNAs are likely involved in many different processes, including genome recognition, early embryonic development, and epigenetic transgenerational inherence [85]. One of the biggest differences between sperm and somatic cells is the fact that somatic cell DNA is wrapped around histones, whereas sperm DNA is tightly packed by protamines, which condenses sperm DNA to one sixth the size of the mitotic chromosomes and carefully protects their DNA [86]. At fertilization, the highly condensed and transcriptionally inert chromatin of the sperm is remodeled into the decondensed and transcriptionally competent chromatin of the male pronucleus [87]. Sperm also carry numerous paternal mRNAs to oocytes at fertilization, facilitating early development [88,89,90]. Sperm is important for the first cell division and can influence the pattern of embryonic gene expression and even phenotypes of the progeny [91]. Epigenetic marks in sperm are extensive and are correlated with developmental regulators [92]. All of the sperm chromatin features are likely to support embryonic development after fertilization. Somatic chromatin does not have such “fine-tuning” for correct embryonic gene expression. Therefore, embryos generated from SCNT often show abnormal reprogramming events compared to fertilized embryos [76], and the cell cycle state of the donor as well as their level of differentiation may be important determinants of reprogramming efficiency. Scientists compared the differences between iPSCs and ESCs and found persistent donor cell gene expression and epigenetic memories in iPSCs [66, 93,94,95,96] however, sperm express fewer genes and carry fewer epigenetic marks than iPSCs. Therefore, the superiority of DSC is the result of sperm chromatin features, which may be useful in embryo development.


The impact of genetic improvement on animal production

While many farmed animals have undergone the process of domestication for millennia, managed selective breeding programs have resulted in striking improvements in productivity. Genetic improvement has resulted in faster, cheaper, healthier, and more-efficient animal production, with reduced impact on the environment. For example, from the 1960s to 2005, selective breeding resulted in 50% larger litter sizes in pigs, an increase of lean pork meat of 37%, and a doubling of lean pork meat per kg of feed intake in chickens, the days to acquire 2 kg of mass reduced from 100 days to 40, the percentage breast meat increased from 12 to 20%, the feed conversion ratio halved, eggs per year increased by 30% and eggs per tonne of feed increased by 80% and finally, in cattle, milk production increased by 67% [13]. These transformative increases in food production represent incredible achievements in just a few decades, albeit the benefits were disproportionately seen in developed countries.

Pedigree-based breeding programmes for major livestock and aquaculture species now routinely incorporate genomic selection, which has been a revolutionary change for selective breeding and food production. Genomic selection [14, 15] involves the use of genome-wide genetic marker data to estimate genomic breeding values (GEBVs) of individuals by means of a genomic prediction equation. This genomic prediction equation is calculated using a ‘training’ or ‘reference’ population where animals have both genotypes and phenotypes, and is then applied to selection candidates, which often have marker genotype information only. The rates of genetic gain have been estimated to lie between 20 and 30% in cattle, pigs, chickens and salmon [16].

Genomic improvements have been accelerated by community-driven pre-competitive research in animal genomics and functional genomics. The major farm-animal genomes have been sequenced [17,18,19], with efforts under way to functionally annotate these genomes to the same standard as the human genome [20,21,22]. Some farm-animal genomes now represent the most contiguous complex genomes ever sequenced [23, 24]. Built on these efforts, genomic tools [25,26,27,28,29,30] and new and cheaper sequencing technologies [31, 32] have been, or will be, major contributors to modern animal breeding and the improved productivity of farmed animals.

Selective breeding is constrained by the standing genetic variation in the species or population of interest, and new variants arising through de novo mutations. Transgenic and genome-editing technologies offer new opportunities for genetic improvement by creating novel beneficial alleles or introducing known desirable alleles from other breeds or species, without the consequences of the linkage drag associated with traditional introgression. Below, we summarize some of the applications of both genetic modification and genome editing to farm-animal productivity and health.


Recent Progress In Downstream Processing

Recent developments in downstream purification processes include the use of high-throughput devices, single-use systems, QbD and PAT, modeling, continuous downstream processing, and integrated continuous downstream processing.

High-Throughput Technologies

High-throughput (HT) technologies have become an important aspect of downstream process development because of their potential to rapidly gather more data related to the process in comparison to traditional laboratory-scale techniques (Benner et al., 2019). Za E coli, HT-compatible bead mills were used for cell disruption (Lazarevic et al., 2013). Various other HT cell disruption/lysis devices have been used, including an 8-well-sonifier for VLPs from E coli, a 24-well-HT sonication device for 15 cells including bacteria, fungi, and yeasts, and microfluidic channels (96-well-format) for thermal treatment, osmotic shocks, and freeze-drying (Baumann and Hubbuch, 2017). In a review, microscale disruption of microorganisms (as low as 200 μl) for parallelized process development was discussed in detail along with their performance compared with high-pressure homogenization (Walther and Dürauer, 2017). HT refolding systems for IB-expressed proteins are also available commercially and are listed in Table 5 (Baumann and Hubbuch, 2017), together with some of the other HTP devices used in downstream process development.

Tablica 5. List of some HTPD systems used for downstream process development (Baumann and Hubbuch, 2017).

HT chromatography systems with different capacities are available (Rege et al., 2006 Coffman et al., 2008 Chhatre and Titchener-Hooker, 2009 Lacki and Brekkan, 2011 𔆬ki, 2012 Chu et al., 2018). Bind and elute evaluations for mAbs and amyloglucosidase have been carried out using pre-packed PreDictor filter plates (GE). Other HT devices have been used successfully, such as AcroPrep Advance 96-well-filter plates (Pall) for the G-CSF, PhyNexus tips (PhyNexus) for Fab fragments, MediaScout MiniChrom columns (Atoll) for mAbs, MediaScout RoboColumn (Atoll) (200� μL columns) for mAb and antibody fragments, and HiTrap columns (GE) for recombinant HIV-1 capsid protein purification (Urmann et al., 2010 Treier et al., 2012 Hung et al., 2013 Muthukumar and Rathore, 2013 Brenac Brochier and Ravault, 2016 Baumann and Hubbuch, 2017). In a recent study, the pairing of MiniColumns and Tecan liquid handlers was used to run up to eight chromatography conditions in one experiment (Benner et al., 2019). An automated HT batch-binding screen using a 96-well-filter-plate (Seahorse Bio) for CEX resins was efficiently optimized for step elution to increase purity and yield for antibodies (McDonald et al., 2016). An HT method based on a microtiter filter plate [96-well with MultiScreenHTS Vacuum Manifold (Merck-Millipore)] was applied to determine the adsorption properties and evaluate the optimal conditions for human serum albumin (HSA) isolation with four MM resins and two IEX resins the findings were verified by laboratory-scale column chromatography (Chu et al., 2018). In another study, an HT process development workflow integrated with a microscale chromatography, DoE, and multivariate data analysis was studied and provided a rational method for screening resins and process parameters (Stamatis et al., 2019). In a study by Andar et al. (2019), a microscale column using IMAC was used for the purification of G-CSF expressed using a cell-free CHO and was compared with a 1 ml IMAC column. A 10-fold decrease in buffer, resin, and time of purification was observed in comparison to conventional columns for similar protein yields. In a recent study, using 96-well-plates containing nickel-functionalized membranes, rapid screening of parameters for membrane protein purification was successfully performed (Feroz et al., 2019). Mixed-mode resins (ionic and hydrophobic interactions) were used in a plate-based HT screening platform for the selection of process parameters to achieve high purity and high overall yield of osteopontin (Guo et al., 2019). A novel microfluidics-based methodology to carry out speedy and multiplexed screening of several MM ligands relative to their potential to bind different target molecules was studied using an artificial mixture (containing IgG and BSA, labeled with different thiol-reactive neutral fluorescent dyes). The study report suggested that this strategy can potentially be utilized as a predictive analytical tool in the context of purification of mAb (Pinto I. F. et al., 2019). The benefits of a HT chromatography system include its availability to predict design space for dynamic binding capacity (DBC), collect data for the prediction of elution behavior, and allow significant investigation using DoE (McDonald et al., 2016). HT chromatography systems have limitations in their potential to reveal the flow distribution of process columns (Singh and Herzer, 2017).

Noyes et al. (2015) studied an ultra scale-down device for high-throughput depth filtration that enabled the parallel assessment of eight single- or multi-layer depth filters (ߠ.2 cm 2 in cross-sectional area). In another study, a novel HT filtration screening system was used to characterize the proteins of different feedstreams with antibody concentrations of up to 20 g/l for their viral filtration performance using either low-interacting or hydrophobically interacting pre-filters. This study indicated the existence of two different fouling mechanisms: an irreversible and a reversible mechanism (Bieberbach et al., 2019). The performance of a pilot-scale TFF system was predicted by devising an ultra scale-down (USD) device consisting of a cell stirred using a rotating disc (2.1 cm 2 of membrane area and 1.7 ml of feed), with good agreement between the USD and TFF devices in terms of the flux and resistance values for a mAb diafiltration stage (Fernandez-Cerezo et al., 2019).

Aqueous two-phase extraction has the potential to selectively separate proteins from unclarified cell culture supernatants directly. In one study, microfluidic aqueous two-phase extraction screening systems with fluorescence microscopy were demonstrated, and it was reported that the partition coefficient (Kstr) measured in PEG 3350–phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) was similar to those calculated for the native protein (São Pedro et al., 2019).

Single-Use Technologies

For single-use or disposable cell harvesting, two main options, namely a single-use centrifuge followed by single-use depth filters or single-use depth filters alone, are used. Single-use depth filters are more common due to the commercial unavailability of single-use large-scale centrifuges (Boedeker et al., 2017). For centrifugation, kSep ® single-use continuous centrifuges (kSep400 and kSep6000S) (Sartorius) were developed and successfully used for cell harvesting to purify recombinant proteins (Mehta, 2014). The Unifuge is another single-use centrifuge available for cell harvesting. Due to the development of single-use filtration techniques, primary and secondary filters can be replaced by a single filtration step. This leads to a lower cycle time, filtration surface area, and buffer requirement. Depth filters can be employed to recover cells from single-use bioreactors up to the 2,000 l scale. However, the number of systems needed for a 2,000 l bioreactor culture is greater, so their use should be analyzed with respect to cost, space, waste, footprint, etc. (Boedeker et al., 2017). The disposable depth filters Stax (Pall), Clarisolve, and Millistak D0HC and X0HC (Merck-Millipore) are available and are used for efficient cell clarification (Schreffler et al., 2015). The depth filters have advantages like ease of scalability, better recovery, consistency, and low cost (Collins and Levison, 2016). However, some issues associated with single-use depth filters include the binding of proteins or DNA (Gupta and Shukla, 2017c).

Single-use chromatography systems can be utilized for the purification of recombinant proteins from culture harvested from an up to 2,000 l bioreactor, depending on product titers, the loading capacity of the column, and process flow rates. Such a system is supported by pinch valves, sensors, and pumps (Boedeker et al., 2017). In a previous study using Protein-A, mixed-mode, and AEX resin columns, single-use continuous purification of mAb was achieved using AKTA periodic counter-current chromatography (Mothes, 2017). It was reported that the per gram mAb operating cost of an SU facility is 22% lower than that of a stainless steel (SS) facility (Gupta and Shukla, 2017c). Single-use TFF systems (Pall and Merck-Millipore) are commercially available for downstream purification, and these systems consist of pumps, pinch valves, a tank, sensors, and tubing manifolds (Boedeker et al., 2017).

Design of Experiments (DoE) Approach

The use of DoE has also been established to increase the performance of downstream process development. In one study, high-pressure homogenization was used to screen critical process parameters (CPPs) using DoE to enhance product titer and achieve adequate product quality, based on predefined critical quality attributes (CQAs) (Pekarsky et al., 2019). A process for the purification of scFv using mixed-mode chromatography was developed using DoE, and it was found that the optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, with the majority of HCPs in the flow-through (Sakhnini et al., 2019). A split DOE approach was successfully used in HIC to remove aggregates, and CEX was used to isolate charge variants and aggregates, resulting in a reduction of the total number of experiments by 25 and 72% compared to a single DoE based on CCD and FFD, respectively (Shekhawat et al., 2019).

Process Analytical Technology (PAT) for Downstream Processing

In downstream processing, PAT tools are used for the analysis of protein concentration, its purity, host cell proteins, host cell DNA, endotoxin, variants (misfolding), and process-related impurities. For these purposes, spectroscopy, spectrometry, HPLC, circular dichroism, and other tools are used to monitor critical quality attributes in chromatography processes. Next-generation sequencing could be used for virus screening, but it is very sophisticated. Further studies are needed to determine the critical points to assure the viral safety of therapeutic proteins (Fisher et al., 2018). One study used an on-line HPLC as a PAT tool for automated sampling of a product stream eluting from a chromatography process column (Tiwari et al., 2018). FTIR spectroscopy as a PAT tool was also used for near real time in-line estimation of the degree of PEGylation in chromatography (Sanden et al., 2019). At-line multi-angle light scattering and fluorescence detectors were used in the downstream processing of HEK293 cell-produced enveloped VLPs containing the HIV-1 Gag protein fused to the Green Fluorescence protein (Aguilar et al., 2019).

Modeling Approach

Modeling and simulations can significantly decrease the number of experiments needed while increasing or collecting experimental data (Hanke and Ottens, 2014). Empirical models are based on a priori identified output data within a defined design space, and mechanistic models are based on physicochemical properties (Baumann and Hubbuch, 2017). Mechanistic modeling is an important process development tool that has been used for chromatography to speed up process development. These models can explain the downstream unit operation at a level of detail that depends on the application (Benner et al., 2019). The use of a mechanistic model of HIC as a PAT tool for pooling decisions to enable aggregate removal for a mAb resulted in higher product purity with respect to offline column fractionation-based pooling (Shekhawat and Rathore, 2019b). An approach toward statistical process control and monitoring of protein refolding during the production of recombinant therapeutic proteins from E coli was described in a study by Hebbi et al. (2019.). This approach used on-line measurements of redox potential, temperature, and pH for the development of a statistical model. This was successfully demonstrated to ensure the quality of the manufactured product consistently. An empirical interpolation (EI) method was used to predict elution performance on a CEX column based on batch isotherm data and revealed good agreement with experimental elution curves for the separation of mAb monomer and dimer mixtures for protein loads up to 40 mg/ml column or about 50% of the column binding capacity (Creasy et al., 2019). Early-stage bioprocess development faces the issues of the definition of optimal operating parameters. Polishing chromatography of a mAb from a challenging ternary feed mixture was optimized by a hybrid approach of the simplex method and a form of local optimization. The findings of the study showed it to be perfectly suitable for the speedy development of bioprocessing unit operations (Fischer et al., 2019). An overview of mechanistic modeling of liquid chromatography was given in a recent study (Shekhawat and Rathore, 2019a).

Continuous Downstream Processing

The shift from a traditional batch process to a continuous process for any product can reduce cost (Schofield, 2018). Systems and techniques for continuous downstream processing of biopharmaceuticals have been developed and used for process development and scale-up. The various technologies of continuous bioprocessing are shown in Figure 4. Continuous centrifugation and TFF-MF are the main methods utilized for cell harvesting or cell removal. Disk stack and tubular bowl centrifuges have been used in continuous operation for the harvesting of a recombinant E coli fermentation that was carried out for a domain antibody production (Voulgaris et al., 2016). A disk-stack continuous centrifuge with periodic and continuous discharge was also used for large-scale clarification of high cell density CHO cell culture for IgG1 mAb production (Richardson and Walker, 2018). The cell lysis techniques (mechanical type) used in continuous mode are high-pressure homogenization and bead milling. In a study by Haque et al. (2016), continuous bead milling was used for the recovery of a recombinant protein, and the process was optimized using RSM.

Slika 4. Flow chart of the different technologies within continuous bioprocessing.

Continuous Precipitation

In the continuous mode of precipitation, a stirred tank reactor, MSMPR, tubular reactor, and centrifugal precipitation chromatography can mainly be used for bioproducts. A continuous precipitation process for mAbs using a tubular reactor was studied with PEG and resulted in 86�% yields with HCP reduction (7,200�,000 ppm) (Hammerschmidt et al., 2016). A coiled flow inverter reactor has also been used for continuous precipitation of clarified cell culture supernatant based on pH, CaCl2, and caprylic acid and resulted in comparable or increased productivity vs. a batch process (Kateja et al., 2016). A combination of reversible cross-linking (ZnCl2) and volume exclusion (polyethylene glycol) agents was also established to continuously precipitate a mAb product in a tubular reactor directly from clarified cell culture fluid (CCCF) (Li Z. et al., 2019).

Continuous Aqueous Two-Phase Extraction

Aqueous two phase extraction in continuous mode can be carried out by column contactors, mixer-settler, spray columns, and rotating disk contactors (Eggersgluess et al., 2014 Espitia-Saloma et al., 2014). A continuous ATPE system for human IgG in a microfluidic device (mixer-settler) in one-stage, multistage, and multistage with recirculation setup was studied with a PEG-3350 phosphate ATPS and resulted in 65 and 90% recovery with one-stage and multistage, respectively, along with 78% in recirculation (Espitia-Saloma et al., 2016).

Continuous Chromatography

In continuous downstream processing, continuous chromatography processes are crucial to achieve high purity for proteins, and these processes are in an advanced stage, with a variety of options. By operating many chromatography columns in a countercurrent or concurrent manner, continuous operation can be achieved, as the loading is carried out in the first column and all of the other steps (washing, elution, regeneration, and re-equilibration) in the subsequent ones (Jungbauer, 2013). Continuous annular chromatography (CAC), simulated moving bed (SMB) chromatography, countercurrent chromatography (CCC), multicolumn countercurrent solvent gradient purification chromatography (MCSGP), and countercurrent tangential (CCT) chromatography are used in the continuous mode of operation (Pagkaliwangan et al., 2018 Rathore et al., 2018 Vogg et al., 2018). Some of the commercially available continuous chromatography platforms are listed in Table 6. One study performing a comparative cost analysis of batch vs. continuous process for 200 kg mAb (annual production) showed that the latter resulted in a decrease in the downstream processing cost of goods (COGs) by ߨ€/g of mAb, with increased requirements of culture medium (Klutz et al., 2016 Somasundaram et al., 2018).

Tablica 6. List of some commercially available continuous chromatography systems used for recombinant biopharmaceuticals.

MCSGP with a four-column system was successfully utilized for the initial capture of an IgG2 mAb from CCCF using CEX with gradient elution (Müller-Späth et al., 2010). A study of the application of CCT chromatography using Protein A resin for the initial capture and purification of two commercial mAbs from CCCF showed that it resulted in similar characteristics in terms of HCP removal, product yield, and purity as conventional column chromatography (Dutta et al., 2015). The use of continuous capture multi-column chromatography (BioSMB) at laboratory scale for a mAb capture process using Protein A resin was successfully validated (Gjoka et al., 2015). In a study on mAb capture using Protein A-based twin-column CaptureSMB, it was reported that the resin cost could be reduced by up to 10�% (Angarita et al., 2015). Twin-column CaptureSMB and three and four-column PCC were studied for capturing mAb using Protein A resin and resulted in similar maximum capacity utilization (Baur et al., 2016). An integrated two-stage chromatographic process platform containing CEX and MM was used for the separation of charge variants and aggregates for three different mAbs, and it was found that the required aggregate (ρ%), HCP (㰐 ppm), and DNA (υ ppb) clearance was achieved (Kateja et al., 2017b). CCT chromatography was also used for a post-capture antibody purification step using MM resins (CEX-HIC) and showed a 5% increase in yield with similar contaminant removal (Dutta et al., 2017). In another study, it was established that the chromatography resin in a two-column continuous system resulted in 2.5-fold more utilization in comparison with single column batch system (Steinebach et al., 2016 Bielser et al., 2018).

In a scale-up study for purification of mAbs, it was reported that buffer savings of around 50% were achieved using a PCC strategy (Angelo et al., 2018). Four different loading scenarios with a Cadence BioSMB MCC for the Protein A mAb capture step were evaluated, and it was concluded that by adding more columns, up to 65% more productivity (at feed concentrations of above 5 g/l) could be achieved (Pagkaliwangan et al., 2018). The effect of particle size (85 vs. 50 μm) on the performance of continuous capture Protein A affinity chromatography was studied with respect to feed titers, load flow rates, and target breakthrough with single column batch, two-column CaptureSMB, and four-column PCC using a DOE approach. The 50 μm resin resulted in better productivity as compared to the 85 μm resin (Baur et al., 2018). The impact of two different quality feeds (one from depth filtration and other from a combination of depth filtration and chromatographic clarification) on Protein A PCC was studied, with the result that there was 49% increased productivity for the chromatographically clarified material over 100 cycles, with 11-fold lower HCP and a 4.4 LRV for HCDNA (El-Sabbahy et al., 2018). Upscaling of Protein A continuous chromatography using the Cadence™ BioSMB PD and the Cadence™ BioSMB Process 80 system was successfully carried out for a 10-day run time using feed from a perfusion culture and resulted in a 400�% increase in vs. batch mode (Ötes et al., 2018). In another study, recovery, and enrichment of the native form of an mAb and of basic and acidic variants were achieved in a multi-column continuous chromatography set-up (three-column) by self-displacement chromatography with a process yield of over 90% (Khanal et al., 2019). In an MCSGP process, by means of the isolation of the main charge isoform of an antibody, the purity was determined by the selection of the product collection window, with negligible influence from the recycle phases (Vogg et al., 2019).

One study considered “standard,” “model-assisted,” and “hybrid” approaches to process characterization for validating continuous twin-column capture chromatography (CaptureSMB) with CCCF containing an IgG4 at 5 g/l (Baur et al., 2019). Methods for the purification of human mAb and their fragments using different chromatography techniques, including continuous chromatography, were also described in a recent study (Ulmer et al., 2019b). A DoE approach using a single column (batch mode) was studied to simulate a multi-column (continuous mode) purification strategy with Protein A capture, anion exchange, and MM cation exchange, and robust and predictable continuous bioprocesses were developed. The process developed yielded total product recovery at or above 74%, HCP (υ ppm) and an aggregate content below 1% (Utturkar et al., 2019).

Continuous Viral Inactivation and Clearance

In the case of manufacturing therapeutics using CHO cells, viral clearance is mandatory (Chiang et al., 2019 Jungbauer, 2019). Viral clearance for mAbs production processes uses a low pH hold because the protein elution occurs at low pH from a Protein A chromatography column. Continuous viral inactivation using a tubular reactor with a static mixer, a coiled flow inverter reactor, and a four-valve system with a mixer has been studied. A fully automatic Cadence™ (Pall) low-pH continuous viral inactivation system was developed and used for virus inactivation (Johnson et al., 2017 Gillespie et al., 2018). A packed-bed continuous viral inactivation reactor was used for the inactivation of two commonly used model viruses with a very low pressure drop and scalability (Martins et al., 2019). In a recent study, a coiled flow inverter was used for continuous low pH viral inactivation, and complete viral inactivation was achieved within the first 14.5 min for both continuous and batch studies (David et al., 2019).

Continuous Refolding

Progress has also been made in the continuous refolding process. A coiled flow inverter reactor, packed column plug flow reactor (incorporated with a mixing system), a CSTR connected with a diafiltration system (for buffer exchange), continuous chromatography systems, or a tubular reactor can be used for continuous refolding. In one study, integrated continuous matrix-assisted refolding and purification by tandem SMB SEC was successfully achieved for Npro fusion proteins expressed in IBs (Wellhoefer et al., 2014). An integrated continuous tubular reactor system was utilized for continuous dissolving, refolding, and precipitation (Pan et al., 2014).

Continuous Formulation

Diafiltration is mainly used for desalting and buffer exchange using ultrafiltration membranes. The co-current and countercurrent modes are used for continuous diafiltration (Kovผs, 2016). In a study by Rucker-Pezzini et al. (2018), continuous three-stage single-pass diafiltration was studied and resulted in buffer exchange of 㺙.75%. A countercurrent staged diafiltration process was performed for continuous protein formulation for a polyclonal IgG with Cadence™ Inline concentrators (Nambiar et al., 2017). Cadence™ in-line concentrators (Delta 30 kDa membranes) were used in the three stages to obtain high conversion in a single pass and provided important insights into the design and operation of a continuous process for antibody formulation (Jabra et al., 2019). Countercurrent dialysis for continuous protein formulation and buffer exchange was done using concentrated solutions of IgG with commercially available hollow fiber dialyzers (1.5 and 1.8 m 2 membrane surface area) (Yehl et al., 2019).

Crystallization for protein formulations can be carried out in continuous mode (Hekmat, 2015 dos Santos et al., 2017 Van Alstine and 𔆬ki, 2018). In one study, continuous crystallization of a full-length therapeutic mAb was carried out using a laboratory-scale stirred tank (with a cooled tubular reactor in bypass) and resulted in a space–time yield of up to 12 g/l.h (Hekmat et al., 2017). Approaches to and the scientific understanding of controls over the crystallization–purification process in continuous crystallization were recently described in a review (Darmali et al., 2019).

A novel concept for the freeze-drying of pharmaceutics in unit-doses was presented by Capozzi et al. (2019), who reported that this configuration made it possible to set up a continuous freeze-drying process.


J.W., L.-H.Y. and C.-B.X. designed the experiments. J.W. performed experiments and data analysis, and wrote the manuscript. Z.-S.Z., L.-H.Y., J.-Q.X., A.A., Y.S., Y.-J.H., G.-Y.W., L.-Q.S., H.T. and Y.L. contributed to assist in performing part of the experiments. S.-M.W., Q.-S.Z., P.Q., Y.-P.W., S.-G.L., C.-Z.M., G.-Q.Z. contributed to part of the field experiments, C.-C.C contributed some rice genetic materials used in this project. C.-B.X. and L.-H.Y. supervised the project and revised the manuscript.

Slika S1. Identification of OsNLP4 knockout mutants and overexpression lines.

Slika S2. Loss-of-function of OsNLP4 results in severe nitrogen deficiency phenotype in DJ background.

Slika S3. OsNLP4 influences plant growth under different ammonium conditions.

Slika S4. OsNLP4 overexpression plants exhibit increased grain yields in the field.

Slika S5. Differentially expressed genes (DEGs) in the WT, ko and OE under low N (LN) and high N (HN) conditions.

Slika S6. OsNLP4 regulates the expression of multiple N metabolism genes in response to nitrate.

Figure S7. OsNLP4 regulates the expression of multiple N metabolism genes in response to ammonium.

Figure S8. Loss-of-function of OsNLP4 significantly affects N metabolism in DJ background.

Figure S9. OsNLP4 regulates ammonium uptake and assimilation.

Figure S10. Expression pattern of OsNLP4 revealed by rice lines expressing OsNLP4 promoter-GUS reporter.

Figure S11. Subcellular localization and protein levels of OsNLP4 in responses to ammonium.

Figure S12. OsNLP4 restores the N deficiency phenotype of Arabidopsis nlp7-1 mutantni.

Figure S13. OsNLP4 broadly regulates the genes related to N utilization and signalling in Arabidopsis nlp7-1 mutantni.

Figure S14. OsNLP4 does not affect the expression of OsNLP1 in response to nitrate and ammonium.

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