Onesposobitev TLR10 v celicah A549 z uporabo tehnologije CRISPR/Cas9

Leskovar, Neža (avtor), Ogorevc, Jernej (mentor)

V genomu bakterij in arhej so prisotne gruče enakomerno prekinjenih kratkih palindromnih ponovitev (CRISPR), ki s proteini Cas tvorijo kompleks. Ta kot del imunskega odziva ščiti organizem pred virusi in plazmidi. Usmerjevalna RNA, ki deluje kot matrica za vezavo na tarčno zaporedje, omogoči endonukleazno delovanje Cas9 na tarčnem mestu, kjer ta povzroči dvojni prelom tarčne DNA ter posledično uničenje invazivnega tujka. V znanosti se CRISPR/Cas9 uporablja za urejanje genomskih zaporedij na točno določenem mestu. Tehnika je zelo uporabna, saj po povzročenem dvojnem prelomu DNA pride do aktivacije endogenih popravljalnih mehanizmov celice, ki pogosto privedejo do naključnih delecij ali insercij in posledično onesposobitve tarčnega gena. Tehnologija CRISPR/Cas9 je učinkovit pristop urejanja genoma za uporabo v raziskovalne namene, kar se je izkazalo tudi pri preučevanju receptorja TLR10. Receptor sodeluje pri imunskem odzivu sesalcev, a njegova funkcija in ligandi še niso natančno opredeljeni. S tehnologijo CRISPR/Cas9 smo uspešno pripravili celično linijo z onesposobljenim TLR10, kar smo potrdili z različnimi metodami in tako postavili osnovo za preučevanje tega receptorja. Pojavlja se vprašanje, ali TLR10 spodbuja ali zavira vnetni odziv imunskega sistema, za kar v literaturi najdemo nasprotujoče si zaključke raziskav. Izvedli smo poskus, pri katerem smo pripravljeno celično linijo z onesposobljenim TLR10 in kontrolne celice z nespremenjenim genomom stimulirali s Pam3Cys ter spremljali izražanje izbranih citokinov s kvantitativno verižno reakcijo s polimerazo v realnem času. Predpostavili smo, da ima receptor protivnetni učinek in bo tako pri celicah, ki ga ne izražajo, prišlo do povečanega izražanja citokinov. V primeru IL8 in IL1β je res prišlo do povečanja izražanja, pri TNFα pa do zmanjšanja. S poskusom smo pokazali uporabnost tehnologije CRISPR/Cas9 za pripravo celičnih linij, ki lahko služijo za funkcionalno anotacijo in pomagajo pri razumevanju delovanja TLR10.

• Jezik: Slovenski jezik
• Ključne besede: tehnologija CRISPR/Cas9, onesposobitev tarčnega gena, receptor TLR10, določitev funkcije TLR10
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-147071
• COBISS.SI-ID: 159899651
• Datum objave v RUL: 22.06.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Generating TLR10 knock-outs in A549 cells using CRISPR/Cas9 technology

Izvleček

Clustered regularly interspaced short palindromic repeats (CRISPR) were found in genomes of bacteria and archaea. With Cas proteins they form a complex working as part of the immune response for protection against viruses and plasmids. Guide RNA represents a matrix for recognition of the target sequence and consequently directs Cas protein activity to the target site where it causes double strand break of DNA and consequently eliminates the invading pathogen. CRISPR/Cas9 is used in research for introducing desired changes to a specific locus in an organism’s genome. Usefulness of the technique is emerging from the fact that the inflicted double strand break in DNA forces the cell to apply an endogenous repair mechanism, which often results in random deletions or insertions and therefore knocks out the target gene. CRISPR/Cas9 technique is a very useful genome editing approach for research purposes which has also been proven when researching TLR10 receptor. This receptor is a part of the immune system in mammals, but its function and ligands have not yet been clearly defined. With CRISPR/Cas9 we successfully developed a cell line with knocked-out TLR10 which we confirmed with different methods. The designed cell line represents a good starting point for understanding the receptor. The main question is whether the TLR10 enhances or represses the inflammatory response of the immune response, which has previously not been clearly answered in the literature due to many contradictory results. We performed an experiment where the knock-out cells and control cells were stimulated with Pam3Cys and expression of selected cytokines was monitored with reverse transcription quantitative real-time polymerase chain reaction. We hypothesized that the TLR10 receptor has an anti-inflammatory effect and therefore the expression of cytokines in knock-out cells will be enhanced. Expression of IL8 and IL1β inflammatory was indeed higher, but the expression of TNFα was lower. With the performed experiment we successfully demonstrated the usefulness of the designed cell line that could be considered as a tool for future studies, which will aid in understanding functions of TLR10.

• Ključne besede: CRISPR/Cas9 technology, gene inactivation, TLR10 receptor, determining the function of TLR10