Recombinant technology has facilitated a significant progress in the field of biopharmaceuticals and has opened new doors for treating various diseases. Biopharmaceuticals are produced in different expression systems, where Chinese hamster ovary cells (CHO) are most commonly used, due to their ease of genetic manipulation and human-like post-translation modifications. The current strategies of cell line development for production of recombinant therapeutics are based on a random integration of the transgene into the CHO genome. With the development of genome editing tools and with draft genomes of several CHO cell lines being available, however, targeted integration into genomic hotspots is becoming an option for generating high-producing and stable cell lines.
For our master thesis project we wanted to find active regions in the CHO genome, which include regulatory elements and sites of transcription. They are known to be in open sites of chromatin, thus allowing transcription machinery to access the DNA. We determined these accessible regions in CHO cells with the ATAC-seq method that probes and fragments DNA with a modified transposase, which inserts sequencing adapters into open regions of chromatin. During optimization, we determined that the optimal number of cells for obtaining a proper library is 5x 104 and we decreased the number of PCR cycles by 2 cycles compared to the protocol in literature in order to get a better complexity of the library. We tested different bioinformatic algorithms and checked three CHO genome references. The reference CHOK1GS_HDv was the most representative since more than 94 % of our reads aligned to it. We tested the reproducibility of our approach on two replicates and concluded that the method is reproducible since we obtained a similar distribution of fragment length and saw a good correlation (Pearson = 0,95) among regions identified in both replicates.
After the successful implementation of the ATAC-seq method on CHO cells we searched for open regions in three CHO parental cell lines at two different time points of the bioprocess (on day 3 and day 7) in order to pinpoint the open regions that are stable and remained open throughout the bioprocess. From those open regions we then identified super-enhancers and compared the results with a publicly available epigenetic study of CHO cells. Among the compared regions, we found that more than half of them really did represent enhancers and concluded that this method is fast, simple, and can be used not only to locate regions of open chromatin, but also to identify super-enhancers.
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