Urejanje gena ALOX 15 z metodo CRISPR/Cas9 v celicah HEK293

Perčič, Anja (author), Narat, Mojca (mentor), Lainšček, Duško (co-mentor)

Vnetje, ki se pojavi brez prisotnosti mikroorganizmov, se imenuje sterilno vnetje. Obstaja več razredov receptorjev, ki so odgovorni za zaznavanje in sprožitev vnetnih odzivov. To so receptorji za prepoznavanje vzorcev oz. motivov na mikroorganizmih (ang. pattern recognition receptors, PRR). Najbolj znani od teh receptorjev so Toll-u podobni receptorji (TLR). Eden izmed njih je TLR4. V primeru sterilnega vnetja, na aktivacijo posredno vpliva tudi encim 15-lipoksigenaza (ALOX 15). Zunajcelični vezikli se ob stresu sproščajo iz celic. Njihova membrana je sestavljena iz fosfolipidne membrane, katere fosfolipidi so substrat za lipidno peroksidacijo. Peroksidira jih ALOX 15. Tako nastajajo hidro(pero)ksilirani fosfolipidi in se lahko vežejo na kompleks TLR4-MD-2, kar povzroči aktivacijo TLR4. Cilj magistrskega dela je bil narediti celično linijo, v kateri se ALOX 15 ne bi izražal. Odločili smo se, da uporabimo sistem CRISPR/Cas9 za modifikacijo genoma. Načrtovali in pripravili smo konstrukt glede na tarčno zaporedje v genomu, ga vnesli v celice in naredili selekcijo s puromicinom. Genomsko modifikacijo smo preverili s testom T7E1, za nadaljnje delo smo izbrali pet klonov, iz teh izolirali DNA in opravili imunodetekcijo s protitelesi za preverjanje izražanja ALOX 15. Uspešno smo naredili celično linijo z manjšim izražanjem ALOX 15, kar nam omogoča nadaljnje raziskovanje mehanizma aktivacije po tej poti.

• Language: Slovenian
• Keywords: molekularna biologija, imunologija, genetika, genom, modifikacije, celične linije, sterilno vnetje, lipoksigenaza, CRISPR
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-113122
• COBISS.SI-ID: 4337544
• Publication date in RUL: 05.12.2019

Secondary language

• Language: English
• Title: Genome editing of ALOX15 using CRISPR/Cas9 in HEK293 cell line

Abstract

Inflammation that occurs in the absence of microorganisms is called sterile inflammation. There are several classes of receptors that are responsible for detecting and triggering inflammatory responses. These are pattern recognition receptors (PRRs). The most famous of these are Toll-like receptors (TLRs), which are transmembrane proteins on the cell surface or in endosomes. One of them is TLR4. In the case of sterile inflammation 15-lipoxygenase (ALOX 15) has a role in the activation of TLR4. Extracellular vesicles (EV) are released from the cell in case of stress. EV have a membrane from lipids that can be substrate for peroxidation. ALOX 15 is the enzyme that is doing the peroxidation of phospholipid. The oxydised lipids can go and bind to the TLR4-MD-2 complex which leads to the activation of TLR4. Since it is not clear how the various mechanisms work, we wanted to make a cell line in which ALOX 15 would not express itself, which was the goal of this master's thesis. We decided to use the CRISPR/Cas9 system. We have designed and prepared the constructs according to the target sequence in the genome, introduced it into cells and we have made selection with puromycin. Genomic modification was verified by the T7E1 assay, five clones were selected for further work. We made a Western blot and immunodetection with antibodies to verify the reduced expression of ALOX 15. We have successfully made a cell line with a lower expression of ALOX 15, which allows further exploring the mechanism of activation along this pathway.

• Keywords: molecular biology, immunology, genetics, genome, modification, cell lines, sterile inflammation, lipoxygenase, CRISPR