Medizinische Universität Graz - Research portal
Selected Publication:
Ledam, R.
Establishing BAP1 iKO AD293 cell lines by using CRISPR/Cas9.
[ Diplomarbeit/Master Thesis (UNI) ] Universität Graz; 2017. pp.95.
FullText
- Authors Med Uni Graz:
- Advisor:
- Heitzer Ellen
- Altmetrics:
- Abstract:
- Heterozygous inactivating germline mutations of BAP1 are associated with a distinct type of melanocytic neoplasm. This autosomal dominant disorder comes along with multiple skin-colored, elevated melanocytic tumors, that show overlapping features with melanoma and are referred to as Wiesner nevi. Most of the melanocytic neoplasms have lost the remaining wild-type allele of BAP1 by further somatic alterations. Due to this, an inducible homozygous BAP1 knock-out model system would help to study the role of BAP1 in human biology and pathology. In this thesis, we report the establishment of BAP1 iKO AD293 cell lines by using CRISPR/Cas9 mediated genome editing. To this end, we used the CRISPR/Cas9 system to introduce double-strand breaks up- and downstream of BAP1 exon 4 and a HDR-mediated repair mechanism, which enabled us to flank BAP1 exon 4 with FRT sites. In a second step, the CRISPR/Cas9 system was used to integrate a (Z)-4-OH-tamoxifen inducible flippase at the AAVS1 locus of the genome, which allows us to induce KO of BAP1 at any given time, simply by the addition of OH-tamoxifen. Flippase-mediated removal of the FRT-flanked BAP1 exon 4 will induce a frameshift in the BAP1 mRNA and will lead to a non-functional, truncated Bap1 protein. In this thesis, we report the successful generation of heterozygous BAP1 iKO AD293 cell lines. By this work, it will be possible to efficiently establish BAP1 iKO iPSC lines in the future. This will help to study the effects of a homozygous BAP1 mutation in different stages of iPSC differentiation into melanocytes and will furthermore help to understand the role of BAP1 in human biology.