title: Cre-mediated site-specific cassette exchange in erythroid cell.
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.
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003850:Deoxyribonuclease I / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D005914:Globins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D006427:Hemin; D005914:Globins; C045073:Cre recombinase; D019426:Integrases; D003850:Deoxyribonuclease I; D004121:Dimethyl Sulfoxide