{"title":"评估两种转座酶通过共转染和超转染方法改善重组蛋白在中国仓鼠卵巢细胞稳定池中的表达。","authors":"Melina Lenser, Hanh Giai Ngo, Lily Sarrafha, Yashas Rajendra","doi":"10.1002/btpr.3496","DOIUrl":null,"url":null,"abstract":"<p><p>Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.</p>","PeriodicalId":8856,"journal":{"name":"Biotechnology Progress","volume":null,"pages":null},"PeriodicalIF":2.5000,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches.\",\"authors\":\"Melina Lenser, Hanh Giai Ngo, Lily Sarrafha, Yashas Rajendra\",\"doi\":\"10.1002/btpr.3496\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.</p>\",\"PeriodicalId\":8856,\"journal\":{\"name\":\"Biotechnology Progress\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2024-07-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnology Progress\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1002/btpr.3496\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Progress","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1002/btpr.3496","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
转座子是一种遗传元件,能够通过转座酶的作用切割和粘贴感兴趣的基因,与随机或定向整合 DNA 相比,它在创建用于重组蛋白质表达的中国仓鼠卵巢(CHO)细胞系方面具有许多优势。独特的转座酶具有不同的识别位点,允许多个转座酶共同转染。它们还允许用第二个转座酶进行超转染(转染到先前转染过的池子或细胞系上),在不破坏先前整合 DNA 的情况下整合同一基因或其他基因的额外拷贝。使用两种独特的转座酶将两种荧光蛋白(EGFP 和 tagRFP657)共转染或超转染到 CHO 细胞中,结果表明,无论基因是共转染还是超转染到现有的稳定池中,它们的表达效率都很高,表达水平(以平均荧光强度衡量)相似。此外,在无 L-谷氨酰胺和有嘌呤霉素的情况下对基因进行双重选择,比单独进行单一选择的表达水平更高。这些结果表明,使用独特的转座酶进行超转染可能是一种有用的策略,可用于提高现有细胞系的滴度或过表达辅助(非治疗)基因,以改善现有池和细胞系的表达和/或产品质量,从而节省大量时间和资源。
Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches.
Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.
期刊介绍:
Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries.
Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.