Inducible expression of coding and inhibitory RNAs from retargetable genomic loci.

Conditional gene expression systems have developed into essential tools for the study of gene functions. However, their utility is often limited by the difficulty of identifying clonal cell lines, in which transgene control can be realized to its full potential. Here, we describe HeLa cell lines, in which we have identified-by functional analysis-genomic loci, from which the expression of transgenes can be tightly controlled via tetracycline-regulated expression. These loci can be re-targeted by recombinase-mediated cassette exchange. Upon exchange of the gene of interest, the resulting cell line exhibits the qualitative and quantitative properties of controlled transgene expression characteristic for the parent cell line. Moreover, by using an appropriate promoter, these cell lines express the tetracycline controlled transcription activator rtTA2-M2 uniformly throughout the entire cell population. The potential of this approach for functional genomics is highlighted by utilizing one of our master cell lines for the efficient microRNA-mediated knockdown of the endogenous human lamin A/C gene.

The use of retroviral vectors for tet-regulated gene expression in cell populations.

Today the treatment of inherited diseases holds a major field in gene therapy, and gamma -retroviral vectors are often the preferred tool for stable introduction of the therapeutic gene(s) into the host cell genome. In many cases, the newly introduced gene has to be constitutively expressed, since enzyme function often is required at all times. However, in some cases gene function might be demanded only transiently, making a strict control of gene expression necessary. For more than a decade, the tet-system has proven to facilitate such strict control by tightly regulating gene expression, thereby assuring high expression levels in almost all organs and tissues. Yet, most of these results were obtained from the analysis of either selected cell clones or transgenic animals. On the contrary, in case of conditional gene expression, as necessary for gene therapy approaches, the use of genetically modified cell populations, where the majority of cells display similar regulatory properties, is required. Therefore, great effort has been undertaken to design viral vectors carrying the response unit that enables homogenous regulation of gene expression in transduced cell populations. This article summarizes critical points that have to be considered for the conditional regulation of gene expression in cell populations mediated by the tet-system. Examples of the required vector elements and tet-system components as well as advice on the handling of the system are given. These tools have been specifically developed to improve population-based gene regulation.

Down-regulation of retroviral transgene expression during differentiation of progenitor-derived dendritic cells.

OBJECTIVE: Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro. MATERIAL AND METHODS: CD34(+) cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis-acting elements. EGFP(+) cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry. RESULTS: Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used. CONCLUSIONS: We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.

Conditional tetracycline-regulated expression of TGF-beta1 in liver of transgenic mice leads to reversible intermediary fibrosis.

Based on the tetracycline-regulated gene expression system, a double-transgenic mouse model for liver fibrosis was established in which the expression of transforming growth factor beta1 (TGF-beta1) can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. TGF-beta1 plasma levels in induced double-transgenic mice reached values ranging from 250 to 1,200 ng/mL, being 10 to 30 times above the normal plasma levels. By applying a cyclic induction-deinduction protocol, deleterious effects of the high plasma TGF-beta1 levels were overcome. By using this protocol, liver fibrosis occurred within a few cycles and progressed further to an intermediary fibrosis when cyclic induction was continued. On histochemical staining, a marked perisinusoidal deposition of extracellular matrix was detected accompanied by the activation of hepatic stellate cells as shown by alpha-smooth muscle actin (alpha-SMA) expression. Apoptosis of hepatocytes was prominent in TGF-beta1 high producers, leading to a decreasing number of TGF-beta1-expressing cells with time. No compensatory proliferation of hepatocytes could be detected. In advanced stages, fibrogenesis could be stopped by switching off TGF-beta1 production and reversal of fibrosis could be shown by (immuno)histochemistry within 6 to 21 days. Determination of messenger RNA (mRNA) levels of procollagen I and III, laminin (B1), matrix metalloproteinase (MMP)-2, -9, and -13, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2 by real-time reverse-transcription polymerase chain reaction (RT-PCR) provided insight into some mechanistic details of the fibrogenic process and its reversal. In conclusion, this model will enable the analysis of fibrogenesis at progressive stages and help in elucidating the cellular changes during development and regression of liver fibrosis caused by elevated TGF-beta1 expression.