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Gewählte Master / Diploma Thesis:

Oliver Putz (2020): Development of a dCas9-ChIP method for targeted enrichment of genomic regions in Chinese hamster ovary cells.
Master / Diploma Thesis - Institut für Tierische Zelltechnologie und Systembiologie, BOKU-Universität für Bodenkultur, pp 71. UB BOKU obvsg

Data Source: ZID Abstracts
Abstract:
Mammalian production systems, like Chinese hamster ovary cells (CHO), have seen an immense growth in recent years, as they are capable of producing complex biopharmaceuticals with human like molecular structures. Although, CHO cells are used extensively for biopharmaceutical production, the organism’s biochemical pathways and genetic regulations have not yet been fully understood, which is crucial for the improvement of cell performance. Importantly, the CHO reference genome is still incomplete due to limitations of conventional next generation sequencing methods. In this study, a chromatin immunoprecipitation (ChIP)-based method is presented, that results in enrichment of specific genomic regions, which can then be sequenced in a targeted approach. A deactivated clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (CRISPR-dCas9) was used in combination with ChIP to target and pull down endogenous or exogenous genomic fragments. The enrichment of the obtained fragments was analyzed with quantitative polymerase chain reaction (qPCR) and their sequences could be determined by Nanopore sequencing. Although, the experiment could not be finished due to the coronavirus pandemic, endogenous and foreign promoter regions were enriched and pulled down successfully. With this method, a known genomic sequence, in the vicinity of a gap in the reference genome, could be targeted and pulled down to determine the unknown gap sequence. Alternatively, this method could be applied for the determination of expression vector integration sites without the need for expensive Whole Genome Sequencing or Targeted Locus Amplification.

Beurteilende(r): Borth Nicole
1.Mitwirkender: Marx Nicolas

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