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doctoral thesis
CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION
Zagreb: University of Zagreb, Faculty of Science, 2023. urn:nbn:hr:217:090828
Mijakovac, Anika
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

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Mijakovac, A. (2023). CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION (Doctoral thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:090828

Mijakovac, Anika. "CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION." Doctoral thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:090828

Mijakovac, Anika. "CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION." Doctoral thesis, University of Zagreb, Faculty of Science, 2023. https://urn.nsk.hr/urn:nbn:hr:217:090828

Mijakovac, A. (2023). 'CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION', Doctoral thesis, University of Zagreb, Faculty of Science, accessed 21 May 2024, https://urn.nsk.hr/urn:nbn:hr:217:090828

Mijakovac A. CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION [Doctoral thesis]. Zagreb: University of Zagreb, Faculty of Science; 2023 [cited 2024 May 21] Available at: https://urn.nsk.hr/urn:nbn:hr:217:090828

A. Mijakovac, "CELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION", Doctoral thesis, University of Zagreb, Faculty of Science, Zagreb, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:217:090828

Metadata
TitleCELL LINES WITH INTEGRATED CRISPRa AND CRISPRi SYSTEM AND THEIR APPLICATION IN ANALYSIS OF IMMUNOGLOBULIN G GLYCOSYLATION
Title (croatian)STANIČNE LINIJE S INTEGRIRANIM CRISPRa I CRISPRi SUSTAVOM I NJIHOVA PRIMJENA U ANALIZI GLIKOZILACIJE IMUNOGLOBULINA G
AuthorAnika Mijakovac
MentorAleksandar Vojta (mentor)
Committee memberVlatka Zoldoš (predsjednik povjerenstva)
Committee memberIvana Ivančić Baće (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2023-03-20, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Universal decimal classification
(UDC)		577 - Biochemistry. Molecular biology. Biophysics
Abstract
Mechanisms regulating immunoglobulin G (IgG) glycosylation are largely unknown. To perform functional validation of genes implicated in IgG glycosylation we developed the HEK293-F system. Utilizing transposons, we integrated dCas9-VPR/dCas9-KRAB expression cassettes in the genome of FreeStyle™ 293-F cells for targeted manipulation (CRISPRa/CRISPRi) of selected genes. Transfection of HEK293-F cells with IgG-gRNAbearing plasmid enabled simultaneous analysis of gene expression and IgG glycome. Utilizing this system, we validated the role of the SPPL3 locus in IgG sialylation. Activation of estrogenregulated genes, RUNX3 and SPINK4, decreased the galactosylated IgG glycoforms, a phenotype characteristic of menopause and aging. To further examine the magnitude of genetic influences on IgG glycosylation in aging we performed a heritability analysis on the glycan clock marker of biological age. Heritability of the glycan clock, when corrected for the age of the individuals, proved to be high (~71%). This served to demonstrate the necessity to develop new technologies able to dissect the observed genetic factors regulating IgG glycosylation associated not only with disease but also with aging.
Abstract (croatian)
Regulatorni mehanizmi glikozilacije imunoglobulina G (IgG) većinom su nepoznati. Kako bismo proveli funkcionalnu potvrdu uloge gena asociranih s IgG glikozilacijom razvili smo sustav HEK293-F. Korištenjem transpozona, ugradili smo kazete dCas9-VPR/dCas9-KRAB u genom stanica FreeStyle™ 293-F za ciljanu manipulaciju (CRISPRa/CRISPRi) odabranih gena. Transfekcija stanica HEK293-F s plazmidom IgG-gRNA omogućila nam je istovremenu analizu ekspresije gena i IgG glikoma. Korištenjem ovog sustava potvrdili smo ulogu lokusa SPPL3 u IgG sijalinizaciji. Aktiviranjem gena reguliranih estrogenom, RUNX3 i SPINK4, došlo je do smanjenja galaktoziliranih IgG glikoformi odnosno pojave fenotipa karakterističnog za menopauzu i starenje. Kako bismo dodatno istražili opseg genetičkih utjecaja na IgG glikozilaciju tijekom starenja, proveli smo analizu nasljednosti markera biološke dobi – glikanskog sata. Nasljednost glikanskog sata se pokazala visokom nakon korekcije za dob sudionika (~71%) čime smo pokazali kako je neophodno razvijati nove tehnologije koje će razjasniti genetičke faktore uključene u regulaciju glikozilacije IgG-a, povezane ne samo s bolestima već i sa starenjem.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:217:090828
Promotion2023
Study programmeTitle: Biology
Study programme type: university
Study level: postgraduate
Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2023-04-03 09:33:13