Retroviral expression in embryonic stem cells and hematopoietic stem cells

SR Cherry, D Biniszkiewicz, L Van Parijs… - … and cellular biology, 2000 - Am Soc Microbiol
SR Cherry, D Biniszkiewicz, L Van Parijs, D Baltimore, R Jaenisch
Molecular and cellular biology, 2000Am Soc Microbiol
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 …
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.
American Society for Microbiology