UC San Diego

Biopharmaceuticals are emerging as a promising avenue for treating a range of diseases 1, and bringing down the costs of production, as well as monitoring the response to treatment, is of high importance. One technology to assist in these needs is next generation sequencing (NGS). In this dissertation, I use emergent NGS techniques to provide valuable resources in two important cell types, the Chinese hamster ovary (CHO) cell-line in Chapter 2, and CD34+ haematopoietic stem and progenitor cells (HSPCs) in Chapter 3. CHO cells are currently the most used cell line for producing recombinant monoclonal antibodies, and optimization of this cell line including media control and gene engineering has brought down production costs 2. However, costs can still be prohibitive, and the genome resources required for novel gene engineering techniques are limited by the resolution of the genome annotation. In Chapter 2, I revise the TSS genome annotation using two different TSS sequencing techniques across multiple tissues in the Chinese hamster. TSSs were detected in 15308 protein-coding genes, and detected TSSs in 13037 of these genes had TSSs revised by at least 10 base pairs from the nearest NCBI RefSeq TSS. More promoter motif elements were detected at the revised TSSs than at the NCBI TSSs. To demonstrate the accuracy and functionality of our revised TSS annotation we activated the dormant Mgat3 gene in CHO cells by CRISPR activation 3 using a novel identified TSS. CD34+ HSPC cells are the target in cytokine therapies to recruit cells to differentiate into desired immune lineages. Studying the clonal lineage multipotent capacity is important for understanding cellular response to therapy. Recent studies have shown that there is heterogeneity in HSPC clones, which are cells from the same phylogenetic origin, in both their growth and hematopoietic multipotent capacity. However, tracking HPSC clones in humans is limited. In Chapter 3, clonal heterogeneity is tracked across multiple donors in steady-state and in response to ex vivo cytokine cocktail using mitochondrial single-cell ATAC-seq. Cells are grouped into clones using naturally occurring somatic mitochondrial mutations, and their lineages assessed using regulatory regions in the nuclear open-chromatin. Larger clones make up a large fraction of donor HSPC donor populations, with no clone preferentially responding to culture. Most clones show multipotent capacity, differentiating into multiple immune lineage progenitors. Overall, this dissertation provides an improved genome annotation in CHO cells and an analysis on HSPC lineage bias in steady-state and in response to cytokine treatment.