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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.

Perez-Campo, F, Spencer, H, Elder, RH, Stern, P and Ward, C 2007, 'Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter.' , Experimental cell research, 313 (16) , pp. 3604-15.

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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.

    Item Type: Article
    Uncontrolled Keywords: Homologous recombination, embryonic stem cells, 5T4 antigen, NEIL2, gene targeting
    Themes: Health and Wellbeing
    Schools: Colleges and Schools > College of Science & Technology
    Colleges and Schools > College of Science & Technology > School of Environment and Life Sciences > Biomedical Research Centre
    Journal or Publication Title: Experimental cell research
    Publisher: Elsevier
    Refereed: Yes
    ISSN: 0014-4827
    Depositing User: RH Elder
    Date Deposited: 06 Oct 2011 16:32
    Last Modified: 20 Aug 2013 18:12
    URI: http://usir.salford.ac.uk/id/eprint/17997

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