Zhenqiu Huang, Hyo-Young Jeong, Luke Nelson, Caitlyn Devine, Guanghua (Benson) Li, Lin Zhang
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引用次数: 0
Abstract
Genetic characterization including Southern blot analysis of recombinant Chinese hamster ovary (CHO) cell lines expressing biologics is an essential prerequisite for regulatory submissions. The transposon-based expression system for CHO cell lines enables the insertion of DNA sequences into the genome and has been widely used by the biopharmaceutical industry. The system yields clones that are both highly productive and genetically stable. However, it often incorporates target genes at multiple locations in the CHO genome, presenting challenges to profile the integration sites using Southern blotting. The standard Southern blot analysis is time-consuming, as multiple rounds of screening for optimal restriction enzymes (REs) are needed to ensure the development of high-quality blots with an optimal resolution of banding patterns. In this study, we developed an innovative in silico Southern blotting tool that leverages the targeted locus amplification (TLA) data and PICRH reference genome to recapitulate only DNA fragments containing the gene of interest (GOI). The tool can plot the size and digestion pattern of these fragments using R packages, allowing for the selection of optimal REs that yield the highest-quality blots virtually, thereby eliminating the need for multiple wet-lab RE screening. The utility of this novel tool has been demonstrated across multiple cell lines and confirmed to be effective and accurate in predicting banding patterns consistent with experimental data. The tool has been implemented in our cell line development workflow which greatly streamlined our cell line genetic characterization.
Biotechnology JournalBiochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍:
Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances.
In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office.
BTJ promotes a special emphasis on:
Systems Biotechnology
Synthetic Biology and Metabolic Engineering
Nanobiotechnology and Biomaterials
Tissue engineering, Regenerative Medicine and Stem cells
Gene Editing, Gene therapy and Immunotherapy
Omics technologies
Industrial Biotechnology, Biopharmaceuticals and Biocatalysis
Bioprocess engineering and Downstream processing
Plant Biotechnology
Biosafety, Biotech Ethics, Science Communication
Methods and Advances.