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Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

Cherry, Sara R. and Biniszkiewicz, D. and van Parijs, L. and Baltimore, D. and Jaenisch, R. (2000) Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells. Molecular and Cellular Biology, 20 (20). pp. 7419-7426. ISSN 0270-7306. http://resolver.caltech.edu/CaltechAUTHORS:CHEmcb00

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Abstract

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.


Item Type:Article
Additional Information:Copyright © 2000, American Society for Microbiology. Received 5 May 2000/Accepted 19 June 2000 The first two authors, S. R. Cherry and D. Biniszkiewicz, contributed equally to this work. We thank members of the Baltimore and Jaenisch labs for advice and discussions. We are grateful to Brian Bates for discussions and comments on the manuscript, George Daly for discussions, Jessie Dausman for blastocyst injections, Ruth Curry for mouse work, and Glen Paradis for FACS sorting. D. Biniszkiewicz was supported by the Deutsche Akademische Austauschdienst. D. Baltimore and R. Jaenisch (NIH/NCI 5-R35-CA44339) are supported by NIH grants. This work was supported in part by the ERC program of the National Science Foundation under award EEC-9843342.
Record Number:CaltechAUTHORS:CHEmcb00
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CHEmcb00
Alternative URL:http://mcb.asm.org/cgi/content/abstract/20/20/7419
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ID Code:3237
Collection:CaltechAUTHORS
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Deposited On:23 May 2006
Last Modified:26 Dec 2012 08:53

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