基因组整合作为稳定高产CHO细胞的一种方法

Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos Pub Date : 1900-01-01 DOI:10.35259/isi.sact.2019_32710

R. L. Kessler, Alexandre Haruo Inoue, Henrique Preti, M. Serpeloni, M. V. Alcantara, F. Marchini, M. Krieger

{"title":"基因组整合作为稳定高产CHO细胞的一种方法","authors":"R. L. Kessler, Alexandre Haruo Inoue, Henrique Preti, M. Serpeloni, M. V. Alcantara, F. Marchini, M. Krieger","doi":"10.35259/isi.sact.2019_32710","DOIUrl":null,"url":null,"abstract":"Methodology: This methodology is based on DNA genomic integration by sleeping beauty transposase. Transfections were made with 25 μg of PEI (polyethylenimine) and 12,5 μg of total DNA (75% of DNA of interest and 25% of transposase DNA) for a total of 107 cells. Two days after, transfectants were selected by FACS, an antibiotic independent process, using GFP as the reporter gene (controled by IRES). Two more sortings rounds were necessary to stablish a 100% transfectant stable population. Cell culture supernatant was collected, and protein of interest was analysed by Western blot and ELISA.","PeriodicalId":427855,"journal":{"name":"Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos","volume":"2 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Genomic integration as a way to stable high producers CHO cells\",\"authors\":\"R. L. Kessler, Alexandre Haruo Inoue, Henrique Preti, M. Serpeloni, M. V. Alcantara, F. Marchini, M. Krieger\",\"doi\":\"10.35259/isi.sact.2019_32710\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Methodology: This methodology is based on DNA genomic integration by sleeping beauty transposase. Transfections were made with 25 μg of PEI (polyethylenimine) and 12,5 μg of total DNA (75% of DNA of interest and 25% of transposase DNA) for a total of 107 cells. Two days after, transfectants were selected by FACS, an antibiotic independent process, using GFP as the reporter gene (controled by IRES). Two more sortings rounds were necessary to stablish a 100% transfectant stable population. Cell culture supernatant was collected, and protein of interest was analysed by Western blot and ELISA.\",\"PeriodicalId\":427855,\"journal\":{\"name\":\"Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos\",\"volume\":\"2 1\",\"pages\":\"0\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1900-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.35259/isi.sact.2019_32710\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.35259/isi.sact.2019_32710","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}

引用次数: 0

摘要

方法学:该方法学基于睡美人转座酶的DNA基因组整合。用25 μg PEI(聚乙烯亚胺)和12.5 μg总DNA(75%的目标DNA和25%的转座酶DNA)转染107个细胞。2 d后，以GFP为报告基因(由IRES控制)，采用不依赖抗生素的FACS方法选择转染物。要建立100%杂交的稳定种群，还需要两轮以上的分选。收集细胞培养上清，用Western blot和ELISA检测感兴趣的蛋白。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Genomic integration as a way to stable high producers CHO cells

Methodology: This methodology is based on DNA genomic integration by sleeping beauty transposase. Transfections were made with 25 μg of PEI (polyethylenimine) and 12,5 μg of total DNA (75% of DNA of interest and 25% of transposase DNA) for a total of 107 cells. Two days after, transfectants were selected by FACS, an antibiotic independent process, using GFP as the reporter gene (controled by IRES). Two more sortings rounds were necessary to stablish a 100% transfectant stable population. Cell culture supernatant was collected, and protein of interest was analysed by Western blot and ELISA.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

Anais do IV International Symposium on Immunobiological e VII Seminário Anual Científico e Tecnológico de Bio-Manguinhos

自引率

0.00%

发文量