Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses
Summary Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero developmen...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
The Company of Biologists
2013-10-01
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| Series: | Biology Open |
| Subjects: | |
| Online Access: | http://bio.biologists.org/content/2/11/1229 |
| _version_ | 1818647146406084608 |
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| author | Adam C. Wilkinson Debbie K. Goode Yi-Han Cheng Diane E. Dickel Sam Foster Tim Sendall Marloes R. Tijssen Maria-Jose Sanchez Len A. Pennacchio Aileen M. Kirkpatrick Berthold Göttgens |
| author_facet | Adam C. Wilkinson Debbie K. Goode Yi-Han Cheng Diane E. Dickel Sam Foster Tim Sendall Marloes R. Tijssen Maria-Jose Sanchez Len A. Pennacchio Aileen M. Kirkpatrick Berthold Göttgens |
| author_sort | Adam C. Wilkinson |
| collection | DOAJ |
| description | Summary
Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2−/− Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability. |
| first_indexed | 2024-12-17T00:57:54Z |
| format | Article |
| id | doaj.art-444509f52e364592a9c151c269f7490d |
| institution | Directory Open Access Journal |
| issn | 2046-6390 |
| language | English |
| last_indexed | 2024-12-17T00:57:54Z |
| publishDate | 2013-10-01 |
| publisher | The Company of Biologists |
| record_format | Article |
| series | Biology Open |
| spelling | doaj.art-444509f52e364592a9c151c269f7490d2022-12-21T22:09:33ZengThe Company of BiologistsBiology Open2046-63902013-10-012111229123810.1242/bio.2013629620136296Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analysesAdam C. Wilkinson0Debbie K. Goode1Yi-Han Cheng2Diane E. Dickel3Sam Foster4Tim Sendall5Marloes R. Tijssen6Maria-Jose Sanchez7Len A. Pennacchio8Aileen M. Kirkpatrick9Berthold Göttgens10 Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Genomics Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS84-171, Berkeley, CA 94720, USA Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide, Seville 41013, Spain Genomics Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS84-171, Berkeley, CA 94720, USA Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Summary Comprehensive analysis of cis-regulatory elements is key to understanding the dynamic gene regulatory networks that control embryonic development. While transgenic animals represent the gold standard assay, their generation is costly, entails significant animal usage, and in utero development complicates time-course studies. As an alternative, embryonic stem (ES) cells can readily be differentiated in a process that correlates well with developing embryos. Here, we describe a highly effective platform for enhancer assays using an Hsp68/Venus reporter cassette that targets to the Hprt locus in mouse ES cells. This platform combines the flexibility of Gateway® cloning, live cell trackability of a fluorescent reporter, low background and the advantages of single copy insertion into a defined genomic locus. We demonstrate the successful recapitulation of tissue-specific enhancer activity for two cardiac and two haematopoietic enhancers. In addition, we used this assay to dissect the functionality of the highly conserved Ets/Ets/Gata motif in the Scl+19 enhancer, which revealed that the Gata motif is not required for initiation of enhancer activity. We further confirmed that Gata2 is not required for endothelial activity of the Scl+19 enhancer using Gata2−/− Scl+19 transgenic embryos. We have therefore established a valuable toolbox to study gene regulatory networks with broad applicability.http://bio.biologists.org/content/2/11/1229ES cellsEnhancerHaematopoiesisTranscription |
| spellingShingle | Adam C. Wilkinson Debbie K. Goode Yi-Han Cheng Diane E. Dickel Sam Foster Tim Sendall Marloes R. Tijssen Maria-Jose Sanchez Len A. Pennacchio Aileen M. Kirkpatrick Berthold Göttgens Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses Biology Open ES cells Enhancer Haematopoiesis Transcription |
| title | Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses |
| title_full | Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses |
| title_fullStr | Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses |
| title_full_unstemmed | Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses |
| title_short | Single site-specific integration targeting coupled with embryonic stem cell differentiation provides a high-throughput alternative to in vivo enhancer analyses |
| title_sort | single site specific integration targeting coupled with embryonic stem cell differentiation provides a high throughput alternative to in vivo enhancer analyses |
| topic | ES cells Enhancer Haematopoiesis Transcription |
| url | http://bio.biologists.org/content/2/11/1229 |
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