{"title":"Cre-mediated site-specific cassette exchange in erythroid cell.","authors":"Xing-Guo Li, Hao-Heng Yan, De-Pei Liu, De-Long Hao, Chih-Chuan Liang","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>Cre-mediated cassette exchange has been developed to perform site-specific chromosomal integration using Cre recombinase. Here, site-specific integration with inverted Lox sites was used to investigate the erythroid cis-acting DNA element in specific chromatin contexts in mouse erythroleukemia cells. Single hygromycin-resistant clones were obtained from the selective semi-solid medium containing hygromycin post-electroporation. PCR and Southern blotting analysis showed single-copy integration of target vector in clones A, B and D. Site-specific cassette exchange was performed in clone A with exchange vector and Cre expression plasmid, followed by gancyclovir selection. Flow cytometry was used for analysis of EGFP gene expression. A 732-bp fragment of human beta-globin gene cluster 5' DNase I hypersensitive site 2 (HS2) was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.</p>","PeriodicalId":21763,"journal":{"name":"Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica","volume":"35 10","pages":"947-51"},"PeriodicalIF":0.0000,"publicationDate":"2003-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Cre-mediated cassette exchange has been developed to perform site-specific chromosomal integration using Cre recombinase. Here, site-specific integration with inverted Lox sites was used to investigate the erythroid cis-acting DNA element in specific chromatin contexts in mouse erythroleukemia cells. Single hygromycin-resistant clones were obtained from the selective semi-solid medium containing hygromycin post-electroporation. PCR and Southern blotting analysis showed single-copy integration of target vector in clones A, B and D. Site-specific cassette exchange was performed in clone A with exchange vector and Cre expression plasmid, followed by gancyclovir selection. Flow cytometry was used for analysis of EGFP gene expression. A 732-bp fragment of human beta-globin gene cluster 5' DNase I hypersensitive site 2 (HS2) was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.