Researcher(s)
- Lucas Tillman, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Developing stable, high-producing Chinese Hamster Ovary (CHO) cell lines remains a major bottleneck in biopharmaceutical manufacturing. Traditional cell line selection methods rely on measuring antibody yield weeks or months into culturing. Unstable lineages are identified late in the process, resulting in significant labor and capital costs. Utilizing barcoded lineage tracing with single-cell RNA sequencing (scRNAseq) offers a promising way to address this by identifying early gene expression patterns that predict long-term clonal stability. My project focuses on building a high-diversity N3WS barcoded plasmid library in E. coli to enable tracking of individual CHO cell lines over time. To construct the library, custom polymerase chain reactions (PCRs) were used to insert variable N3WS barcode sequences into an antibody-expressing vector. Custom electroporation protocols were leveraged to insert the library into E. coli with high efficiency. However, sequencing data from initial transformations revealed that a significant portion of the plasmids were assembled incorrectly or were missing a barcode entirely. Ongoing work centers on troubleshooting the PCR and Gibson Assembly protocols. By refining reaction conditions, the goal is to optimize assembly so transformed constructs are consistently accurate before scale up to a full library generation. After assembly is optimized, next steps involve generating a diverse barcoded library, verifying barcode diversity through amplicon sequencing, and transfecting the library into CHO cells. Ultimately, samples will be collected for scRNAseq and analyzed to link clonal lineages to early transcriptional signatures.