magistrska naloga
Urša Nagode (Avtor), Borut Štrukelj (Mentor), Matjaž Vogelsang (Komentor)

Povzetek

Biološka zdravila so spremenila način in uspešnost zdravljenja mnogih bolezni zaradi njihovega tarčno specifičnega delovanja. Za pravilno zvitje proteina in ustrezne posttranslacijske spremembe je pomembna premišljena izbira ekspresijskega sistema, v katerem bo potekala proizvodnja želenega rekombinantnega proteina. Na tem področju je v ospredju (poleg prokariontskih celic bakterije Escherichia coli, ki se uporabljajo za bioprodukcijo enostavnejših neglikoziliranih proteinov) uporaba sesalskih celičnih linij, kjer prevladujejo ovarijske celice kitajskega hrčka (celice CHO). Slednje imajo sicer vse potrebne karakteristike za proizvajanje biološko aktivne oblike proteina, a v zadnjem času raste želja po izboljšanju celičnih linij za dosego višjih titrov in boljše kvalitete produkta. Tu nastopijo različne metode za urejanje genoma, ki temeljijo na prelomih dvojne vijačnice in s tem povzročijo želene genetske spremembe. Trenutno je najbolj atraktivna tehnologija CRISPR/Cas9 (skupki regularno porazdeljenih kratkih palindromskih ponovitev in z njimi povezani protein 9), s pomočjo katere številni avtorji poskušajo izbiti določene gene ali pa povečati njihovo izražanje. V okviru magistrske naloge smo zato poskusili implementirati tehnologijo CRISPR/Cas9 v razvoj celičnih linij CHO z namenom, da bi potencialno lahko ustvarili visoko producirajoče celične linije. Za modelne gene smo izbrali PlekhB1, Rps6KA2, Sept1, Flt1 in Fkbp10, kateri naj bi imeli vpliv na nižjo produktivnost celic. Najprej smo na podlagi rastne krivulje za uporabljeno celično linijo CHO-der3 določili mejo celične gostote za precepljanje celic (6,0 x 106 viabilnih celic/ml) in razmerje precepljene kulture (1/12) ter s tem omogočili ohranjanje celic v eksponentni fazi rasti. Za ločevanje transficiranih od netransficiranih celic in učinkovito delovanje sistema CRISPR/Cas9, smo določili optimalne selekcijske pogoje (5 μg/ml puromicina, 5 dni) in dosegli 50 – 60 % uspešnost pri izbijanju gena PlekhB1 ob uporabi vektorja kot dostavnega sistema. Ugotovili smo, da je sistem CRISPR/Cas9 deloval izven tarčno z enako učinkovitostjo kot na tarčnem mestu gena PlekhB1. Ob sočasni dostavi 8 različnih sgRNA za gene PlekhB1, Rps6KA2, Sept1 in Flt1 (2 sgRNA za vsak gen) smo dosegli 10 – 20 % uspešnost izbitja posameznega gena. Pri dostavi sgRNA v obliki PCR-pomnožkov ali v obliki vektorjev ni bilo razlik v učinkovitosti. Na koncu smo z dostavo sgRNA v obliki vektorjev izbili gene PlekhB1, Rps6KA2, Sept1, Flt1 in Fkbp10 z visoko (50 – 94 %) Implementacija tehnologije CRISPR/Cas v razvoj celičnih linij CHO učinkovitostjo. Ob primerjavi titrov kontrolnih klonov in klonov z izbitim genom nismo zaznali statistično značilne razlike med skupinama. Zaključimo lahko, da smo tehnologijo CRISPR/Cas9 uspešno implementirali v razvoj celičnih linij CHO za tarčno izbijanje posamičnih genov in pokazali, da geni PlekhB1, Rps6KA2, Sept1, Flt1 in Fkbp10 nimajo vpliva na produktivnost celic.

Ključne besede

celična linija CHO;tehnologija CRISPR/Cas;puromicin;produktivnost celic;tarčno izbijanje genov;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [U. Pavlič]
UDK: 576.3:615.32(043.3)
COBISS: 4543857 Povezava se bo odprla v novem oknu
Št. ogledov: 398
Št. prenosov: 66
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Implementation of the CRISPR/Cas technology in CHO cell line development
Sekundarni povzetek: Biopharmaceuticals have changed the way and success of treating many diseases due to their target specificity. In order for a protein to have proper folding and the right post-translational modifications, the expression system in which the recombinant protein will be produced needs to be thoughtfully selected. Mammalian cell lines are the leading system (aside from the prokaryotic cells of Escherichia coli bacteria used for production of simpler non-glycosylated proteins) for use in the mentioned field, among which Chinese hamster ovary cells (CHO cells) are the first choice. Although they have all the necessary characteristics for producing biologically active forms of proteins, the need for improvement of these cell lines grows nowadays in order to achieve higher titers and product of better quality. At this point different methods for genome editing, that induce double stranded breaks and result in desired genetic modifications, come in consideration. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and CRISPR associated protein 9) technology is currently the most promising of the aforementioned methods, which is why many authors are using it to knockout or overexpress certain genes. In the scope of this master thesis we tried implementing the CRISPR/Cas9 technology in CHO cell line development to potentially establish cell lines with increased productivity. We used PlekhB1, Rps6KA2, Sept1, Flt1 and Fkbp10 for our model genes, because they were linked with impact on lowering the cell productivity. Firstly, on the basis of the established growth curve for the CHO-der3 cell line used in our experiments, we determined the cell density value for subculturing cells (6,0 x 106 viable cells/ml) and cell splitting ratio (1/12) in order to maintain cells in the exponential growth phase. To separate the transfected cells from the non-transfected ones and to achieve efficient editing with the CRISPR/Cas9 system, we set the optimal selection conditions (5 μg/ml of puromycin for 5 days) and obtained efficiencies of 50 – 60 % for knocking out the PlekhB1 gene by sgRNA-expressing plasmid delivery. We have discovered that the CRIPSR/Cas9 system off target efficiency was the same as on the target site for the PlekhB1 gene. With simultaneous delivery of 8 sgRNAs for PlekhB1, Rps6KA2, Sept1 and Flt1 genes (2 sgRNAs for each gene) we achieved efficiencies of 10 – 20 % for knocking out individual genes. Between delivering the sgRNA with PCR-amplicons or plasmids there were no differences in the indel frequency observed. At the end we efficiently (50 – 94 %) knocked out PlekhB1, Rps6KA2, Sept1, Flt1 and Fkbp10 genes by sgRNA-expressing plasmid delivery. We then compared the titers of control clones and clones with knocked out genes but we didn't discover any statistically significant differencies between the two groups. In conclusion, we showed that CRISPR/Cas9 technology can be successfully implemented in CHO cell line development for targeted knock-out of specific genes, and demonstrated that PlekhB1, Rps6KA2, Sept1, Flt1 and Fkbp10 genes have no effect on the cell productivity.
Sekundarne ključne besede: Biološka zdravila;Celična biologija;
Vrsta dela (COBISS): Magistrsko delo/naloga
Komentar na gradivo: Univ. Ljubljana, Fak. za farmacijo
Strani: X, 59 f.
ID: 12040862