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dc.contributor.authorPerez-Campo, Flor-Maria
dc.contributor.authorSpencer, Helen L
dc.contributor.authorElder, Rhoderick H
dc.contributor.authorStern, Peter L
dc.contributor.authorWard, Christopher M
dc.date.accessioned2009-06-30T10:56:45Z
dc.date.available2009-06-30T10:56:45Z
dc.date.issued2007-10-01
dc.identifier.citationNovel 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.en
dc.identifier.issn0014-4827
dc.identifier.pmid17765223
dc.identifier.doi10.1016/j.yexcr.2007.07.021
dc.identifier.urihttp://hdl.handle.net/10541/71917
dc.description.abstractThe 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.
dc.language.isoenen
dc.subject.meshAnimals
dc.subject.meshAntigens, Neoplasm
dc.subject.meshBase Sequence
dc.subject.meshBiological Markers
dc.subject.meshCell Differentiation
dc.subject.meshCell Line
dc.subject.meshClone Cells
dc.subject.meshEmbryonic Stem Cells
dc.subject.meshGenetic Vectors
dc.subject.meshGreen Fluorescent Proteins
dc.subject.meshMice
dc.subject.meshMolecular Sequence Data
dc.subject.meshPromoter Regions, Genetic
dc.subject.meshRecombination, Genetic
dc.subject.meshThymidine Kinase
dc.titleNovel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter.en
dc.typeArticleen
dc.contributor.departmentStem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK.en
dc.identifier.journalExperimental Cell Researchen
html.description.abstractThe 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.


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