Konstrukcija TALEN-a za inaktivaciju gena HNF1A u stanicama HepG2

master's thesis

• Title (english): TALEN construction for knockout of HNF1A gene in HepG2 cells
• Author: Mihaela Tremski
• Mentor: Aleksandar Vojta (mentor)
• Committee member: Aleksandar Vojta (predsjednik povjerenstva)
• Committee member: Damjan Franjević (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2016-02-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Tehnologija genomskog inženjerstva omogućava specifičnu i efikasnu modifikaciju u genomu domaćina za razliku od prijašnjih metoda mutageneze pomoću rekombinaza, virusnih integraza i transpozaza. Kimerna nukleaza TALEN je fuzija proteina TALE i FokI nukleaze. TALEN vezujućom domenom specifično prepoznaje ciljnu sekvencu DNA, a nukleazna domena uvodi dvolančani lom. Popravak ovakvih lomova NHEJ mehanizmom često rezultira malim delecijama ili insercijama koje dovode do pomaka u okviru čitanja te inaktivacije ciljanog gena. Cilj ovog rada bio je konstrukcija TALEN-a za ciljno mjesto u genu HNF1A i transfekcija u HepG2 stanice. TALEN-i su uspješno konstruirani metodom Golden Gate kloniranja i transfecirani u HepG2 stanice. Detekcijom fluorescencije proteina EGFP utvrđena je umjerena efikasnost transfekcije. Proveden je T7E1 test koji nije dovoljno osjetljiv za detekciju mutanata u HepG2 stanicama potvrđene efikasnosti transfekcije. Za dobivanje stabilne stanične linije s inaktiviranim genom HNF1A treba provesti selekciju na linijama dobivenim uzgojem od pojedinačnih stanica. Stanična linija s inaktiviranim HNF1A bila bi izvrstan model za istraživanja funkcije gena HNF1A.

Abstract (english)

Genomic engineering technology enables specific and efficient modification of the genome of the host cell, as opposed to previous mutagenesis methods using site-specific recombinases, viral integrases and transposases. The chimeric nuclease TALEN is the result of a fusion of the TALE protein and the FoxI nuclease. TALEN's protein-binding domain specifically recognizes a target DNA sequence and the nuclease domain introduces a double-strand break. Repair by NHEJ often results in small deletions or insertions which cause frameshift mutations and inactivation of targeted genes. The aim of this study was to design a TALEN constructs specific for the target location in the HNF1A gene and transfect it into HepG2 cells. TALENs were successfully constructed by the Golden Gate cloning method and transfected into HepG2 cells. Moderate transfection efficiency was identified after fluorescence microscopy of EGFP. The T7E1 assay was not sensitive enough to detect knock out mutants in HepG2 cells for which efficient transfection was confirmed. Stable cell lines with knockout HNF1A could be obtained by selection of cell lines derived from single cells. The cell line with inactivated HNF1A gene would represent an excellent model for studies of HNF1A gene function.

• Keywords: genomsko inženjerstvo; TALEN; inaktivacija gena; HNF1A; HepG2 stanična linija; transfekcija
• Keywords (english): genomic engineering; TALEN; knock out; HNF1A gene; HepG2 cell line; transfection
• Language: croatian
• URN:NBN: urn:nbn:hr:217:229537
• 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
• Created on: 2017-05-12 11:41:03