TALEN construction for knockout of the HNF1A gene in HepG2 cells

Tremski, Mihaela (2016) TALEN construction for knockout of the HNF1A gene in HepG2 cells. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

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.

Item Type: Thesis (Diploma thesis)
Keywords: genomic engineering, TALEN, knock out, HNF1A gene, HepG2 cell line, transfection
Supervisor: Vojta, Aleksandar
Co-supervisor: Markulin, Dora
Date: 2016
Number of Pages: 64
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 24 Mar 2016 11:45
Last Modified: 24 Mar 2016 11:45
URI: http://digre.pmf.unizg.hr/id/eprint/4612

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