Cell Line Development
Stable, high-producing cell lines are fundamental when producing therapeutic antibodies at the scale needed to fulfill global demand. Cell line engineering holds immense potential for rapid cell line development. Genome editing with site-specific, programmable nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas nucleases offer tremendous potential in cell line development. They also produce site-specific DNA double-strand breaks (DSBs). DSBs can be repaired by imprecise, error prone non-homologous end joining (NHEJ) or by homology directed repair (HDR) pathways, leading to targeted mutagenesis. However, the often low (<5%) frequency of gene edits hinders detection of these edits by gel- or sequencing-based methods or requires labor-intensive clonal isolation and expansion.
The successful development of antibody therapeutics also hinges on the genetic stability of the chosen cell lines. Here, early decisions and assessments are critical for establishing quality and safety over time. Limitations in precision for gene copy number assessment can cause uncertainty during characterization and expansion, which can lead to wasted time and resources. Technologies that can generate reproducible results with great precision are key to successful cell line development.
Benefits of Droplet Digital PCR (ddPCR)
Droplet digital PCR (ddPCR) technology offers the sensitivity, accuracy, and absolute quantification needed for gene edit confirmation in cell line development. It can reproducibly determine gene vector copy number to assess the stability and safety of cell lines to assure productivity. Also, ddPCR complements the custom cell engineering workflow as it allows scientists to develop a screening strategy to characterize the expected frequency of homologous recombination. This is achieved by distinguishing between clones harboring homozygous and heterozygous edits. In addition, designing ddPCR Genome Edit Detection Assays is easily done for any target of interest using Bio-Rad's easy-to-use Digital PCR Assay site. This enables a much more rapid and efficient workflow and provides the ability to quickly identify and focus on desired clones.
Learn How Bio-Rad’s ddPCR Technology Helps Cell Line Development Efforts
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Systematic Quantification of HDR and NHEJ Reveals Effects of Locus, Nuclease, and Cell Type on Genome-Editing. Miyaoka Y. et al. Sci Rep. 2016 Mar 31;6:23549. DOI: 10.1038/srep23549. PMID: 27030102; PMCID: PMC4814844.
Transcriptome and Proteome Analysis of Steady-State in a Perfusion CHO Cell Culture Process. Bertrand V. et al. Biotechnol Bioeng. 2019 Aug;116(8):1959-1972. DOI: 10.1002/bit.26996. Epub 2019 May 7. PMID: 30997936.
Learn how ddPCR provides high sensitivity, quantitative reporting with high reproducibility.
Discover an ultra-sensitive, ddPCR-based method for detecting HDR and NHEJ alleles produced by CRISPR/Cas9 and TALEN genome editing systems.