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

2.50
Hdl Handle:
http://hdl.handle.net/10541/71917
Title:
Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter.
Authors:
Perez-Campo, Flor-Maria; Spencer, Helen L; Elder, Rhoderick H; Stern, Peter L; Ward, Christopher M
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.
Affiliation:
Stem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK.
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
Issue Date:
1-Oct-2007
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
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
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.en
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

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