engleski

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

Genome editing tools, such as TALEN (transcription activator-like effector nuclease) or CRISPR-Cas9 (CRISPR- associated protein-9 nuclease) systems, enable functional studies by targeted gene knockout. They introduce double- stranded breaks (DSBs) into a DNA molecule in a sequence- specific manner, thereby stimulating the error-prone non- homologous end joining repair mechanism, leading to probable gene inactivation when the coding sequence is targeted. Vectors for expression of TALEN and Cas9-based constructs targeting the human IL6ST and HNF1A genes were assembled and tested for their ability to introduce DSBs when transfected into cultured cells using the luciferase assay. The Cas9- based construct targeting the IL6ST gene was shown to be active, while the two TALEN- based constructs did not introduce DSBs above background level. Both the TALEN and the CRISPR-Cas9 constructs targeting the HNF1A gene were found to be active, with the TALEN showing higher activity in a dose- dependent manner. The constructed genome-editing tools can be used for functional analysis of the putative role of HNF1A and IL6ST genes in IgG glycosylation, as shown previously by genome wide association studies.

gene knockout ; TALEN ; CRISPR ; HNF1A ; IL6ST ; genome editing

nije evidentirano