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    Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter.

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    Authors
    Perez-Campo, Flor-Maria
    Spencer, Helen L
    Elder, Rhoderick H
    Stern, Peter L
    Ward, Christopher M
    Affiliation
    Stem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK.
    Issue Date
    2007-10-01
    
    Metadata
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    Abstract
    The use of gene mutation/knock-out strategies in mouse embryonic stem (ES) cells has revolutionized the study of gene function in ES cells and embryonic development. However, the construction of vectors for homologous recombination strategies requires considerable expertise and time. We describe two novel vectors that can generate site specific knock-out or EGFP knock-in ES cells within 6 weeks from construct design to identification of positive ES cell clones. As proof-of-principle, we have utilized the knock-out targeting vector to modify the NEIL2 locus in ES cells. In addition, using the knock-in vector, we have inserted EGFP downstream of the 5T4 oncofetal antigen promoter in ES cells (5T4-GFP ES cells). Undifferentiated 5T4-GFP ES cells lack EGFP and maintain expression of the pluripotent markers OCT-4 and NANOG. Upon differentiation, EGFP expression is increased in 5T4-GFP ES cells and this correlates with 5T4 transcript expression of the unmodified allele, loss of Nanog and Oct-4 transcripts and upregulation of differentiation-associated transcripts. Furthermore, we demonstrate that fluorescent activated cell sorting of 5T4-GFP ES cells allows isolation of pluripotent or differentiated cells from a heterogeneous population. These vectors provide researchers with a rapid method of modifying specific ES cell genes to study cellular differentiation and embryonic development.
    Citation
    Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter. 2007, 313 (16):3604-15 Exp. Cell Res.
    Journal
    Experimental Cell Research
    URI
    http://hdl.handle.net/10541/71917
    DOI
    10.1016/j.yexcr.2007.07.021
    PubMed ID
    17765223
    Type
    Article
    Language
    en
    ISSN
    0014-4827
    ae974a485f413a2113503eed53cd6c53
    10.1016/j.yexcr.2007.07.021
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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