The nuclear distribution of Polycomb during Drosophila melanogaster development shown with a GFP fusion protein.

The chromatin protein Polycomb (PC) is necessary for keeping homeotic genes repressed in a permanent and heritable manner. PC is part of a large multimeric complex (PcG proteins) involved in generating silenced chromatin domains at target genes, thus preventing their inappropriate expression. In order to assess the intranuclear distribution of PC during mitosis in different developmental stages as well as in the germ line we generated transgenic fly lines expressing a PC-GFP (Green Fluorescent Protein) fusion protein. Rapidly dividing nuclei were found to display a rather homogeneous PC-GFP distribution. However, with increasing differentiation a pronounced subnuclear pattern was observed. In all investigated diploid somatic tissues the bulk of PC-GFP fusion protein is depleted from the chromosomes during mitosis: however, a detectable fraction remains associated. In the male germ line in early spermatogenesis, PC-GFP was closely associated with the chromosomal bivalents and gradually lost at later stages. Interestingly, we found that PC is associated with the nucleolus in spermatocytes, unlike somatic nuclei. In contrast to mature sperm showing no PC-GFP signal the female germ line retains PC in the germinal vesicle.

Control of transgene expression using local hyperthermia in combination with a heat-sensitive promoter.

BACKGROUND: Local production of therapeutic proteins, e.g. for cancer treatments, is based on gene therapy approaches and requires tight spatial and temporal control of gene expression. Here we demonstrate the use of local hyperthermia of varying intensity and duration to control the expression of a transgene under control of the thermoinducible hsp70 (heat shock protein) promoter. METHODS: Heat-induced expression of the EGFP (green fluorescent protein) reporter gene was characterized using a stably transfected glioma C6 cell line expressing the EGFP gene under control of the heat inducible minimal hsp70 promoter both in vitro and in vivo for subcutaneous tumors in immunodeficient mice. RESULTS: A heat shock of 20-30 min at 43 degrees C in cell culture led to a maximum EGFP concentration at about 24 h. Heat treatments at higher temperature (up to 48 degrees C) but with shorter durations (down to 30 s) also induced strong EGFP expression. Local heating in situ led to gradients in EGFP expression which decreased with increasing distance from the heat source. CONCLUSION: Local hyperthermia, in combination with a heat sensitive promoter, represents a method for the spatial and temporal control of transgene expression.

Spatial and temporal control of transgene expression in vivo using a heat-sensitive promoter and MRI-guided focused ultrasound.

BACKGROUND: Among the techniques used to induce and control gene expression, a non-invasive, physical approach based on local heat in combination with a heat-sensitive promoter represents a promising alternative but requires accurate temperature control in vivo. MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control allows automatic execution of a predefined temperature-time trajectory. The purpose of this study was to demonstrate temporal and spatial control of transgene expression based on a well-defined local hyperthermia generated by MRI-FUS. METHODS: Expression of the green fluorescent protein (GFP) marker gene was used. Two cell lines were derived from C6 glioma cells. The GFP expression of the first one is under the control of the CMV promoter, whereas it is under the control of the HSP70 promoter in the second one and thus inducible by heat. Subcutaneous tumours were generated by injection in immuno-deficient mice and rats. Tumours were subjected to temperatures varying from 42 to 50 degrees C for 3 to 25 min controlled by MRI-FUS and analyzed 24 h after the heat-shock. Endogenous HSP70 expression and C6 cell distribution were also analyzed. RESULTS: The results demonstrate strong expression at 50 degrees C applied during a short time period (3 min) without affecting cell viability. Induced expression was also clearly shown for temperature in the range 44-48 degrees C but not at 42 degrees C. CONCLUSIONS: Heating with MRI-FUS allows a tight and non-invasive control of transgene expression in a tumour.