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Cre-mediated site-specific cassette exchange in erythroid cell.

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  • 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences Peking Union Medical College, Beijing 100005, China.

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.

PMID:
14515215
[PubMed - indexed for MEDLINE]
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