diplomsko delo
Greta Junger (Author), Gregor Gunčar (Mentor)

Abstract

Kinazi podoben protein mešanega rodu, krajše MLKL, je protein s pomembno vlogo pri nekroptozi, vrsti programirane celične smrti z morfološkimi značilnostmi nekroze. Pri nekroptozi sodeluje tako, da tvori oligomer, ki se vgradi v membrano in s tem povzroči celično smrt. Veliko je že znanega o samem poteku in vlogi MLKL proteina pri nekroptozi, nekoliko manj pa o dejanski strukturi oligomera, ki pri tem sodeluje. Za sledenje proteinu MLKL je bil že pripravljen konstrukt M33-mCherry. M33 je nanotelo, ki je specifično za N-konec človeškega MLKL, mCherry pa je fluorescenčni protein. Skupaj tvorita kromotelo. V okviru diplomske naloge smo pogledali, koliko časa po transfekciji s pcDNA3.1-M33-mCherry pride do največjega izražanja kromotelesa M33-mCherry. Transficirali smo celice HEK293 Flp-In, ki imajo v svojem genomu stabilno vstavljen inducibilen zapis za MLKL. Izražanja proteina MLKL nismo inducirali. V različnih časovnih razmakih po transfekciji smo celice lizirali in dobljene lizate analizirali s poliakrilamidno gelsko elektroforezo ob prisotnosti NaDS ter s prenosom western. Po 16 h in 48 h smo celice analizirali tudi s fluorescenčnim mikroskopom, s čemer smo preverili, če in kje v celici se fuzijski protein izraža. Ugotovili smo, da je bila uspešnost izvedene transfekcije nizka, da pa se je protein tam, kjer je bil prisoten, večinoma lokaliziral le v delu celice. Izraženost M33-mCherry se v prvih 24 h ni bistveno spremenila, opazno pa je bilo povečanje po 48 h. Glede na rezultate prenosa western smo sklepali, da je prišlo do delne razgradnje fuzijskega proteina M33-mCherry. Ker transfekcija v našem primeru ni bila najuspešnejša, in ker delamo s celicami Flp-In, ki imajo možnost stabilne vstavitve poljubnega gena v celični genom, bi lahko v nadaljevanju v celice poleg zapisa za MLKL stabilno vstavili še zapis za M33-mCherry, kar bi olajšalo nadaljnje eksperimente.

Keywords

nekroptoza;protein MLKL;M33 nanotelo;fluorescenčni protein mCherry;kromotelo;Flp-In;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [G. Junger]
UDC: 577.112(043.2)
COBISS: 117138179 Link will open in a new window
Views: 70
Downloads: 25
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Other data

Secondary language: English
Secondary title: Expression of M33-mCherry protein in HEK293-Flp-In cell line with integrated MLKL gene
Secondary abstract: Mixed lineage kinase domain-like pseudokinase (MLKL) is a protein than plays a vital role in necroptosis, a type of programmed cell death with morphological characteristics of necrosis. When phosphorylated, MLKL oligomerises, translocates to the cell membrane and causes cell death. A lot is already known about the process of oligomerisation; however, the structure of oligomer and the mechanism of its action remain to be determined. To learn more about MLKL and its mechanism, a fusion protein M33-mCherry, composed of M33 nanobody and red fluorescent protein mCherry in a pcDNA3.1 vector, had already been prepared. M33 is specific for the N terminal region of human MLKL, and together with mCherry forms a chromobody. In our study, we wanted to determine at what time, after transfection, the expression of a chromobody is the most prominent. First, we transfected HEK293 Flp-In cells that already have integrated MLKL, however, we did not induce its expression, as that would result in cell death. At certain time stamps after transfection, the cells were lysated and analysed, using SDS-PAGE and western blot. After 16 h and 48 h, the cells were also observed under a fluorescent microscope to determine the transfection efficiency and the localisation of chromobody inside the cell. Results showed that overall, the transfection efficiency was very low, despite some cells having high chromobody expression levels. In those cells, M33-mCherry mostly localised in a specific part of the cell. The results of western blot showed that the chromobody was partially degraded. We also concluded that the expression of chromobody did not change significantly in the first 24 h after transfection, however, there was an increase after 48 h. Future experiments could be optimised by exploiting the Flp‑In system, which allows for a stable integration of the desired DNA sequence at a specific genomic location. By stably integrating M33-mCherry, transfection could be avoided altogether, which would make future experiments easier to conduct.
Secondary keywords: M33 nanobody;mCherry;chromobody;MLKL;Flp-In;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 35 str.
ID: 15849479