Tet-system for the regulation of gene expression during embryonic development

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTA ^CMV (M1) and rTA ^CMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and -galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTA ^CMV (M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTA ^CMV -3/NZL-2 embryos at E13.5. Doxycycline abolished -gal expression in tTA ^CMV (M1)/NZL-2 but induced it in rTA ^CMV -3/NZL-2 embryos including late stages of embryogenesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that double reporter animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.     

Transgenic expression of Angiopoietin 1 in the liver leads to changes in lymphatic and blood vessel architecture

To investigate the possible role of the Angiopoietins in vessel remodelling, we overexpressed one of the angiopoietins, Angiopoietin-1 (Ang1), in the hepatocytes of mice by means of the conditional binary transgenic system. Animals were examined by Doppler ultrasound, and dissected livers were analyzed by immunohistochemical staining. Double transgenic mice presented with enlarged spleens and kidneys, enlarged, disorganized blood vessels located near the surface of the liver, sprouting, dilation, and disorganization of liver lymphatics, and turbulent flow in about 1/4 of the blood vessels sampled. Most of these characteristics completely resolved within 12 weeks of turning off the expression of the Ang1 transgene, illustrating a dependence on the continual presence of Ang1 for maintenance of the vascular phenotype. Conditional Angiopoietin-1 overexpression in the liver of mice leads to a phenotype highly reminiscent of portal hypertension illustrating that Ang1 can drive both vascular and lymphatic vessel remodelling and may play a role in portal hypertension.     

Gene therapy on demand: Site specific regulation of gene therapy

Since 1990 when the first clinical gene therapy trial was conducted, much attention and considerable promise have been given to this form of treatment. Gene therapy has been used with success in patients suffering from severe combined immunodeficiency syndromes (X-SCID and ADA-deficiency), Leber's congenital amaurosis, hemophilia, β-thalassemia and adrenoleukodystrophy. Last year, the first therapeutic vector (Glybera) for treatment of lipoprotein lipase deficiency has been registered in the European Union. Nevertheless, there are still several numerous issues that need to be improved to make this technique more safe, effective and easily accessible for patients.     

Conditional Transgene Expression in Endothelial Cells

The ability to tightly control transgene expression in vivo provides an opportunity to determine the role of certain gene products at different times during development and/or in response to different stimuli. We have characterized and evaluated a tetracycline-responsive endothelial-specific binary system during mouse development, by engineering several transgenic lines which drive the expression of a tetracycline- controlled transactivator (tTA) under the control of either the Tek or Tie promoters (driver lines). We have also generated a responder line which carries multiple copies of the tTA DNA binding element (tetos) upstream of a reporter gene coding for a nuclear targeted -galactosidase (responder lines). No expression of the target transgene was detected in mice homozygous for the reporter transgene. On mating the driver lines with the responder line, expression of -galactosidase from the reporter transgene was detected within the endothelium. Responder transgene expression was repressed rapidly upon addition of doxycycline to the drinking water. Importantly, this repression was reversible upon withdrawal of the drug. This approach should be useful to deliver the expression of potentially toxic gene products or rescue embryonic mutations that affect either the endothelial lineage or production of growth factors which are secreted systemically.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.     

A new mouse model for temporal‐ and tissue‐specific control of extracellular superoxide dismutase

The extracellular isoform of superoxide dismutase (EC‐SOD, Sod3) plays a protective role against various diseases and injuries mediated by oxidative stress. To investigate the pathophysiological roles of EC‐SOD, we generated tetracycline‐inducible Sod3 transgenic mice and directed the tissue‐specific expression of transgenes by crossing Sod3 transgenic mice with tissue‐specific transactivator transgenics. Double transgenic mice with liver‐specific expression of Sod3 showed increased EC‐SOD levels predominantly in the plasma as the circulating form, whereas double transgenic mice with neuronal‐specific expression expressed higher levels of EC‐SOD in hippocampus and cortex with intact EC‐SOD as the dominant form. EC‐SOD protein levels also correlated well with increased SOD activities in double transgenic mice. In addition to enabling tissue‐specific expression, the transgene expression can be quickly turned on and off by doxycycline supplementation in the mouse chow. This mouse model, thus, provides the flexibility for on–off control of transgene expression in multiple target tissues. genesis 47:142–154, 2009. © 2009 Wiley‐Liss, Inc.     

Spatial and temporal down-regulation of transgene expression using the TRSID-silencer in mice: application to Prnp

Spatial and temporal control of ovine prion protein (Prnp) gene expression was achieved in mice using two transgenes: a Prnp minigene with tet-operator sequences inserted 5' to exon 1 and a mouse neurofilament genomic clone carrying the chimeric-repressor TRSID cDNA. In bi-transgenic mice, ovine PrP(C) expression could be reversibly controlled in neuronal cells by doxycycline treatment whereas it remains constant in other cell types. Overall, this model opens opportunities to assess the involvement of cell types in prion diseases and PrP physiological function. It demonstrates the potentiality of the TRSID-silencer to precisely control temporal and spatial gene expression in vivo.