Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells

Abstract

(18,19,31). In particular, the lack of significant provirus transcription in ES cells and their differentiated descendants has hampered the use of retroviral vectors in transgenic experiments (5,12,32). Interestingly, this block in provirus expression is maintained upon differentiation of infected cells despite the fact that primary infection of cells after differentiation results in efficient expression (6,7,26).Transcriptional repression is thought to be mediated by both cis-acting de novo methylation of the integrated proviruses and cell-type-specific trans-acting transcriptional repressors (5, 9, 23). The effect of trans-acting factors on retroviral expression through binding of specific sequences within the promotors of retroviruses has been examined in many studies (29,30,35). In fact, the mouse stem cell virus (MSCV) long terminal repeat (LTR) was generated by the modification of the sequences within the LTR to increase the affinity for positive factors and decrease the affinity for negative regulators (20).In contrast, the role of methylation in silencing has been less clear. DNA methylation is thought to be a general mechanism used by cells to silence foreign DNA and may be involved in the cell defense against transposable elements (39). DNA methylation has also been associated with the repression of gene expression and the silencing of viral control elements (2,14,38). Exogenously introduced retroviruses silenced in vitro and in vivo can be reactivated by treatments that result in genomewide demethylation. In addition, transcriptionally silent endogenous retroviral elements are reactivated upon loss of genomic methylation in Dnmt1 knockout mice (38). Therefore, DNA methylation is thought to causally repress expression of retroviral promoters in a variety of cell types.ES cells provide a good model to study the role of DNA methylation in retroviral silencing. First, it was demonstrated that ES cells have high de novo methylation activity, which leads to effective methylation of integrated retroviral vectors, while little or no de novo methylation activity was detected in differentiated cells (21). In addition, ES cells were genetically modified to alter the endogenous level of DNA methylation by the targeted disruption of the maintenance methyltransferase gene Dnmt1. ES cells homozygous for this mutation proliferate normally with their genomic DNA highly demethylated, while differentiated cells and mice die due to the loss of genomic methylation (21,22). Therefore, these modified ES cells are useful to study the effect of DNA methylation on retroviral gene expression. In addition, ES cells can be induced to differentiate in vitro or in vivo, allowing the study of DNA methylation and its effect on long-term expression.Both Moloney virus-based and MSCV-based retroviral vectors have been used for gene transduction in a variety of cells. The MSCV vector is different from the typical Moloney virus vector in that the mutations in the LTR have allowed expression in a larger host range (8,20). To this end, we ...

Efficient lentiviral transduction of Herpesvirus saimiri immortalized T cells as a model for gene therapy in primary immunodeficiencies

“…We demonstrated that eGFP detection in HVS-T cell lines transduced with HRSIN-CSGW represents vector transduction (and not pseudotransduction 16 or expression from nonintegrated vectors 17,18 ) since the percentage of eGFP þ cells remained stable for at least 48 days as shown in Figure 2. This also supports that gene silencing 19 is not occurring in lentiviral-transduced HVS-T cells.…”

“…17,18 Additionally, the expression of the transgene by transduced cells can be blocked by gene silencing. 19 The expression of eGFP protein was determined on days 7, 28 and 48 after a single round of cell transduction on day one. Figure 2 shows that the high percentage of transduced cells …”

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

“…39,40 This indicates significant differences in epigenetic transgene regulation between primitive embryonic and adult stem cells, or, in general, between pregastrulation and postgastrulation cells. 41 It will be interesting to determine whether the type of control regions present in the vector, which lacked any element known to affect chromatin modulation besides a strong retroviral enhancer/promoter, 26 contributed to the promising outcome after immunoselection. Previous studies in murine HSCs used vectors with different cis-acting elements.…”

Evidence implicating the candidate schizophrenia/bipolar disorder susceptibility gene G72 in mitochondrial function

“…The reason for this low-frequency variegated pattern of expression is unknown, but it may be related to cytogenetic instability and karyotypic variability of this cell line 12,13 and also possibly to the unusual genomic structure of the G72 gene, in particular its location within a genomic region enriched in Long Terminal Repeat Elements 6 which are prone to transcriptional silencing. 14 In addition to…”