prikaz prve stranice dokumenta Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint
Embargo period 2025-02-21
master's thesis
Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint
Zagreb: University of Zagreb, Faculty of Science, 2023. urn:nbn:hr:217:941693
Tintor, Erna
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Tintor, E. (2023). Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:941693

Tintor, Erna. "Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:941693

Tintor, Erna. "Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint." Master's thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:941693

Tintor, E. (2023). 'Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint', Master's thesis, University of Zagreb, Faculty of Science, accessed 23 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:941693

Tintor E. Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2023 [cited 2024 December 23] Available at: https://urn.nsk.hr/urn:nbn:hr:217:941693

E. Tintor, "Uspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:217:941693

Metadata
TitleUspostava ljudskih staničnih linija s endogeno eksprimiranim proteinom tubulinom fluorescentno obilježenim metodom CRISPaint
Title (english)Establishment of human cell lines with endogenous expression of protein tubulin fluorescently tagged using the CRISPaint method
AuthorErna Tintor
MentorIva Marija Tolić (mentor)
MentorMaja Matulić (komentor)
Committee memberMaja Matulić (predsjednik povjerenstva)
Committee memberBiljana Balen (član povjerenstva)
Committee memberPetra Peharec Štefanić (član povjerenstva)
Committee memberTomislav Ivanković (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2023-02-21, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Abstract
Za promatranje procesa unutar žive stanice, većinom se koristi fluorescencijska mikroskopija. Glavni zahtjev fluorescencijske mikroskopije je to da željeni predmet proučavanja u stanici mora fluorescirati. Jedan od načina za promatranje stanice pod fluorescencijskim mikroskopom je da se stanični proteini obilježe fluorescentnim proteinima. Međutim, egzogeno unešene sekvence za fuzionirane proteine generiraju proteine s neprirodnom razinom ekspresije, a endogeni protein ostaje neobilježen. Endogeno obilježavanje staničnih proteina s fluorescentnim proteinima jedan je od načina za njihovu vizualizaciju bez utjecaja na jačinu ekspresije. Tehnologija CRISPR-Cas (engl. Clustered Regularly Interspaced Short Palindromic Repeats) često je korištena u genetičkom inženjerstvu za jednostavno i precizno uređivanje genoma. U ovom radu je korištena novo razvijena metoda CRISPaint (engl. CRISPR-assisted insertion tagging). To je sustav koji uz pomoć tehnologije CRISPR-Cas omogućava ugradnju gena za fluorescentni protein u genom tj. u ciljanu sekvencu gena, da bi se proizveo endogeni protein obilježeni fluorescentnim biljegom. Optimizirala sam protokol za transfekciju uz pomoć plazmida sustava CRISPaint-a i uspostavila sam dvije ljudske stanične linije, HeLa i RPE1, s endogeno obilježenim proteinom tubulinom sa zelenim fluorescentnim proteinom. Ove stanične linije se dalje mogu koristiti za promatranje stanične diobe pod fluorescencijskim mikroskopom, a metoda CRISPaint se može primijeniti za endogeno obilježavanje i drugih proteina od interesa.
Abstract (english)
Fluorescence microscopy is used to observe processes inside a living cell and its main requirement is that the desired object of study in the cell must fluoresce. One way to observe a cell under a fluorescence microscope is to label cellular proteins with fluorescent proteins (FP). However, exogenously introduced sequences for fusion proteins produce proteins with unnatural expression levels while the endogenous protein remains unlabeled. Endogenous labeling of cellular proteins with FP is one way to visualize them without affecting the strength of expression. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) technology is today often used in genetic engineering to simply and precisely edit the genome. In this work, the newly developed method CRISPaint (CRISPR-assisted insertion tagging) was used. It is a system that, using CRISPR-Cas technology, enables the incorporation of a gene for FP into the genome in order to produce an endogenous fluorescently tagged protein. I optimized the transfection protocol using CRISPaint system plasmids and established two human cell lines, HeLa and RPE1, with endogenously labeled protein tubulin with green FP. These cell lines can be used to observe cell division under a fluorescence microscope and the CRISPaint method can be applied for endogenous labeling of other proteins.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:941693
Study programmeTitle: Molecular Biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
Type of resourceText
File originBorn digital
Access conditionsEmbargoed access
Embargo expiration date: 2025-02-21
Terms of useLicence In copyright icon
Created on2023-03-01 13:21:21