Uspostava rekombinantnih mišjih embrionalnih matičnih stanica u svrhu istraživanja fleksibilnosti DNA

master's thesis

• Title (english): Establishment of recombinant mouse embryonic stem cells for use in DNA flexibility assays
• Author: Ivan Mikičić
• Mentor: Francis Stewart (mentor)
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-02-19, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Kvantitativnim mjerenjem fleksibilnosti DNA u stanicama HEK293 metodama mjesno-specifične rekombinacije i Southern blota uočeno je da je fleksibilnost DNA in vivo dvostruko veća nego in vitro, što je pripisano namatanju DNA oko nukleosoma te kromatinskoj remodelaciji. Utišavanje razvojno-specifičnih gena lokalnim remodeliranjem kromatina također bi moglo uzrokovati promjenu fleksibilnosti DNA. Cilj ovog diplomskog rada bio je pripremiti mišje embrionalne matične stanice (mEMS) s ugrađenim različitim genskim kazetama za mjerenje i usporedbu fleksibilnosti DNA s mišjim neuralnim matičnim stanicama (mNMS). Kloniranjem gena proizvedena je serija od 10 plazmida koji sadrže različite genske kazete: mjesta loxP različitih udaljenosti (od 70 bp do 2359 bp), 5' i 3' homologiju s kromosomskim genom mNanog te gen za otpornost na antibiotik blasticidin. Kloniranjem gena razvijene su probe za provjeru ispravnosti kazeta metodom Southern blot, te su razvijene početnice za provjeru metodom lančane reakcije polimerazom (PCR). Uvođenjem dvolančanog loma potpomognutim metodom CRISPR-Cas9 uspješno su ugrađene kazete svih 10 plasmida u gen mNanog, što je potvrđeno metodom Southern blot.

Abstract (english)

Using quantitative measurement of DNA flexibility in HEK293 cells by methods of site-specific recombination and Southern blot, it has been observed that in vivo DNA flexibility is double that of in vitro DNA, which has been ascribed to nucleosomal DNA wrapping and chromatin remodeling. Silencing of development-specific genes by local chromatin remodeling could also cause a change in DNA flexibility. The aim of this Master's thesis was to prepare the mouse embryonic stem cells (mESCs) with different integrated gene cassettes for measurement and comparison of DNA flexibility with mouse neuronal stem cells (mNSCs). Gene cloning was used to prepare a series of ten plasmids containing the different gene cassettes: loxP sites of different distances (from 70 bp to 2359 bp), 5' and 3' homology to chromosomal mNanog gene and blasticidin resistance gene. Probes for cassette verification by Southern blotting were produced by gene cloning and primers for verification by polymerase chain reaction (PCR) were designed. Introducing a double-stranded break assisted by CRISPR-Cas9 method, all 10 targeting constructs were successfully inserted into the mNanog gene, which was verified by Southern blotting.

• Keywords: fleksibilnost DNA; remodeliranje kromatina; mišje embrionalne matične stanice; neuralne matične stanice; Cre-loxP; CRISPR-Cas9; Southern blot
• Keywords (english): DNA flexibility; chromatin remodeling; mouse embryonic stem cells; neural stem cells; Cre-loxP; CRISPR-Cas9; Southern blot
• Language: croatian
• URN:NBN: urn:nbn:hr:217:642635
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2019-10-19
• Created on: 2018-03-14 12:43:14