Next-Generation Sequencing-Based In Silico Transgene Integration Profiling Tool Accelerates Cell Line Genetic Characterizations

IF 3.2 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Zhenqiu Huang, Hyo-Young Jeong, Luke Nelson, Caitlyn Devine, Guanghua (Benson) Li, Lin Zhang
{"title":"Next-Generation Sequencing-Based In Silico Transgene Integration Profiling Tool Accelerates Cell Line Genetic Characterizations","authors":"Zhenqiu Huang,&nbsp;Hyo-Young Jeong,&nbsp;Luke Nelson,&nbsp;Caitlyn Devine,&nbsp;Guanghua (Benson) Li,&nbsp;Lin Zhang","doi":"10.1002/biot.202400675","DOIUrl":null,"url":null,"abstract":"<div>\n \n <p>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.</p>\n </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 3","pages":""},"PeriodicalIF":3.2000,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Journal","FirstCategoryId":"5","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/biot.202400675","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
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.

下一代基于测序的硅转基因整合分析工具加速细胞系遗传表征
表达生物制剂的重组中国仓鼠卵巢(CHO)细胞系的遗传表征包括Southern blot分析是提交监管文件的必要先决条件。基于转座子的CHO细胞系表达系统能够将DNA序列插入基因组中,并已广泛应用于生物制药行业。该系统产生的克隆既高产又遗传稳定。然而,它通常在CHO基因组的多个位置整合靶基因,这给使用Southern blotting分析整合位点带来了挑战。标准的Southern blot分析是耗时的,因为需要多轮筛选最佳限制性内切酶(REs),以确保开发具有最佳条带模式分辨率的高质量印迹。在这项研究中,我们开发了一种创新的硅Southern印迹工具,该工具利用靶向位点扩增(TLA)数据和PICRH参考基因组来概括含有目标基因(GOI)的DNA片段。该工具可以使用R包绘制这些片段的大小和消化模式,允许选择产生最高质量印迹的最佳RE,从而消除了多次湿实验室RE筛选的需要。这种新工具的实用性已经在多个细胞系中得到证明,并证实在预测与实验数据一致的条带模式方面是有效和准确的。该工具已在我们的细胞系开发工作流程中实施,大大简化了我们的细胞系遗传表征。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Biotechnology Journal
Biotechnology Journal Biochemistry, 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.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信