Novel Tag-and-Exchange (RMCE) Strategies Generate Master Cell Clones with Predictable and Stable Transgene Expression Properties

JOURNAL OF MOLECULAR BIOLOGY

卷:390

Article

Qiao, Junhua¹  Oumard, Andre¹  Wegloehner, Wolfgang²  Bode, Juergen^1,3 

[1] Helmholtz Ctr Infect Res, Dept Mol Biotechnol Epigenet Regulat, D-38124 Braunschweig, Germany

[2] InVivo Biotech Serv GmbH, D-16761 Hennigsdorf, Germany

[3] Hans Borst Ctr Heart & Stem Cell Res, MHH, Hannover Med Sch, D-30625 Hannover, Germany

关键词: site-specific recombination;    RMCE;    targeted integration;    tag and exchange;    gene expression;   

DOI  :  10.1016/j.jmb.2009.05.012

来源: Elsevier

【 摘 要 】

Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is clean in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (similar to 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype. (C) 2009 Elsevier Ltd. All rights reserved.

【 授权许可】

Free