Conversion of adult pancreatic a-cells to beta-cells after extreme beta-cell loss

Nature 2010 Vol. 464: 1149-1154

In diabetes, insulin-producing beta-cells of the pancreas are destroyed (Type-1 Diabetes) or dysfunctional (Type-2 Diabetes) resulting in abnormally high blood glucose levels eventually leading to severe organ damage. New ways to find a cure for diabetes are under investigation and comprise:

• generation of beta-cells from ES-cells
• replication of existing beta-cells
• conversion of other cells into functional beta-cells

In a study recently published in Nature, Herrera and colleagues used transgenic mice expressing the Diphteria Toxin (DT) receptor under control of the rat insulin promoter to specifically ablate beta-cells. Following DT treatment, 99% of the beta-cells in adult mice were destroyed. However, functional beta-cells regenerated slowly in all pancreatic islets in the following months. To determine whether these new insulin producing cells emerge from other sources than pre-existing beta-cells, Herrera and colleagues carried out careful cell lineage tracing studies. Using an elaborate system of transgenic mice carrying cells that were labeled before beta-cell ablation, they first showed that the increase in beta-cell mass does not result from increased beta-cell proliferation but from regeneration of non-beta cells. In the following detailed analysis the Tet-On Advanced System was instrumental in determining that regenerated beta-cells where derived from glucagon-producing alpha-cells. Thus when almost all beta-cells are destroyed but alpha-cells are spared, functional beta-cells emerged through transdifferentiation of alpha-cells. The findings by Herrera and colleagues may provide new approaches for diabetes therapies.

The present publication provides another powerful example of ‘recycling’ previously established transgenic Tet mice when generation of mice carrying multiple transgenes is required to address complex scientific questions. In this context the continuously growing ‘zoo’ of Tet transgenic mice, accessible to academic scientists through repositories like The Jackson Labs, EMMA and Riken, will further facilitate the use of the Tet Technology to perform breakthrough science.

Direct conversion of fibroblasts to neuronal cells by defined factors under Tet-control

Nature 2010 Jan 27 (Epub ahead of print)

A new highlight in cellular reprogramming was recently published in Nature. It was previously shown that a combination of four transcription factors was sufficient to reprogram mouse and human fibroblasts to a pluripotent state. Now scientists from Stanford University were able to convert mouse fibroblasts directly into functional neurons bypassing this pluripotent state.

In the publication highlighted here, Wernig, Südhof and colleagues asked whether expression of neural-lineage-specific transcription factors could directly turn skin cells into neurons. To this end they infected mouse embryonal fibroblast with a lentiviral pool of Tet-controlled neuronal transcription factors. They identified a subset of 5 factors sufficient for conversion of fibroblasts into neuronal cells as determined by morphological traits and electrophysiological recordings of membrane properties. In subsequent experiments they evaluated whether induced neuronal (iN) cells could even be derived from postnatal cells. To address this question they isolated fibroblasts from 3 day old mice of lines expressing the Tet-On transactivator. Lentiviral transductions of these fibroblasts with the previously identified, Tet-controlled transcription factors revealed that the same set of factors was sufficient to derive iN (induced neuronal) cells from postnatal cells. Further characterization of the relative contribution of each of the five factors showed that a single factor "Ascl1" is critical to induce some neuronal traits and that expression of 2 other additional factors (Brn2 and Myt1l) facilitates neuronal conversion and maturation.

These findings mark a breakthrough in biomedical stem cell science. It may well be possible to convert any "adult" cell type into other lineages thus opening up new perspectives for regenerative medicine.

Uncovering Early Response of Gene Regulatory Networks in ESCs by Systematic Induction of Transcription Factors

Gene regulation networks determine cellular identity and destiny. Alterations to these interactive networks result in cellular differentiation processes but also pathological developments. Understanding the interactions of the roughly 2000 transcription factors encoded in the mouse genome is challenging but key towards our understanding of the mechanisms involved in these regulatory biological systems.
M.S.H. Ko and collaborators dared to tackle this tremendous task and have made first inroads applying the Tet system for the systematic and reversible perturbation of the TF network. In a recent publication they describe the generation of embryonic stem cell (ESC) lines, each equipped with one of 50 different transcription factors under control of the Tet gene switch. All mutant ESC lines were generated by inserting individual FLAG tagged transcription factor genes into one defined tetracycline controllable locus (ROSA-Tet-locus, Masui et al., 2005). This locus allows the reproducible and tightly controlled expression of the inserted TF gene, which can be easily introduced by recombinase mediated cassette exchange. Induction of the respective transcription factor in each ES cell line allows the comparative study of the resulting network perturbations under analogous conditions. 
Initial microarray studies comparing induced versus non-induced state in these ES cell lines revealed clear differences between various TFs in terms of relative impact on the perturbations in the global transcriptional network. One of the genes with an exceptionally strong effect on the transcriptome was the caudal type homeobox 2 gene (Cdx2). Cdx2 over-expressing cells were subsequently used in a detailed proof of principle study demonstrating the utility of the described ESC lines. An impressive amount of methods was applied to characterize the effect of Cdx2 induction in the stem cells comprising ChIP-qPCR analysis for the identification of DNA binding sites and FLAG immunoprecipitation combined with mass spectrometric analysis to isolate and identify protein binding partners.
While this is only the first insight into these complicated networks, the tetracycline inducible ES cell lines established and initially characterised by Ko and colleagues in the present publication provide a fundamental resource for reverse engineering biological networks not only in ES cells but also in differentiated cells or even in animals models.

Development of repressible sterility to prevent the establishment of feral populations of exotic and genetically modified animals  

In a recent publication in "Aquaculture", R. Dunham and collaborators present first results on a novel method that allows commercial breeding  as shown for catfish and oysters but renders them reproductively non-viable in the wild. The method relies on the identification of species-specific developmental switches which are "knocked-out" using inducible expression of antisense or ds- constructs. In catfish, the authors combined a stage specific promoter driving expression of the Tet-Off transactivator controlling expression of antisense or double stranded transcripts directed against BMP2 which is known to be crucial during early embryonic development. In oysters, HOXCG1 / HOXCG3 genes were targeted which encode transcription factors specific for oyster larval development. In both species, tTA-dependent down-regulation of the target gene resulted in induced mortality. Embryos that were maintained in the presence of doxycyline showed significantly lower mortality, sometimes comparable to control groups that were treated with unspecific DNA.
Animals escaping from commercial farms endanger wild populations and the surrounding marine ecosystem.  While the data are far from turning this method into a soon to be implemented practical solution, they hold promise that a similar approach with improved strategies (e.g. gene switch under Tet-On control; non-antibiotic inducers, etc.) may prevent or at least significantly reduce the environmental risks of "fish farming" in the not too distant future.

Inducible expression of coding and inhibitory RNAs from retargetable genomic loci

The utility of regulatable expression systems is often limited by the difficulty to identify those clonal cell lines which allow the intended efficient control of the gene of interest. Weidenfeld et al. have addressed this problem and established a RMCE (recombinase-mediated cassette exchange) based system for the generation of stable tetracycline-inducible cell lines with predictable expression characteristics. To achieve this goal, the authors functionally identified genomic loci in a new HeLa-rtM2 cell line which warrant a homogeneous and highly dynamic tetracycline response. A HygTk cassette was subsequently placed in these loci, allowing for efficient Flp recombinase mediated exchange of expression vectors. The RMCE mediated insertion of Tet-controlled genes of interest reproducibly preserved the quantitative and qualitative properties of the parent cell line from the first screen. The potential of this approach as an expedient tool in functional genomics was highlighted by its application to the efficient, microRNA-mediated downregulation of endogenous genes.

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

Virus-free induction of pluripotency and subsequent excision of reprogramming factors

Nature 2009 Mar 1. [Epub ahead of print]

Inducible pluripotent stem cells (iPS) are generated by activating genes in adult cells to "reprogram" them and, consequently, have many characteristics of embryonic stem cells. So far their therapeutic use seems limited because established reprogramming technologies are based on viral delivery of reprogramming factor transgenes, raising safety concerns for patients treated with such cells.
Two groups of scientists (Woltjen et al. and Kaji et al.) report in this month`s Nature on a new way to transform human fibroblasts into iPS cells.  In the first publication, Nagy and colleagues used the piggyBac transposon to introduce the four transcription factors sufficient to generate iPS cells. In order to achieve temporal expression control, reprogramming factors were placed behind a Tet-On responsive promoter. Stable iPS cells expressed various characteristic pluripotency markers and, most interestingly, piggyBac insertions allowed seamless removal of reprogramming factors after their expression had become dispensable.
Both simplified and virus independent methods of iPS cell generation will further accelerate the understanding of the reprogramming process and may open up new ways for clinical approaches to regenerative medicine.

Seeding and propagation of untransformed mouse mammary cells in the lung

Science 2008 Vol 321 (5897): pp 1841-1844

Since many years the metastatic dissemination of cancer cells has been proposed to occur at a very late stage of tumor development. It was thought that mutations or changes in expression levels accumulate in tumor cells over time before they are able to evade from their primary location. Recently this paradigm is more and more put into question by data suggesting that the metastatic process may begin earlier than previously thought. This assumption is now supported by work from Podsypanina et al. published in Science 321, 1841 (2008). The authors manipulated mouse mammary cells, so that the timing of myc and ras oncogene expression is experimentally controlled by the Tet Technology and injected them into the bloodstream of mice. The cells were able to establish residence in the ectopic environment of the lung and remained clinically undetectable in the absence of oncogene expression. After Doxycycline induced activation of the introduced oncogenes the cells developed into metastatic pulmonary lesions.
These results strongly suggest that metastatic dissemination is not limited to the final phase of primary tumor development, but might occur much earlier. Aside from their significance for our understanding of the mechanisms of cancer metastasis these results could directly impact strategies for cancer therapy in the near future.

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.

09/14/2008
Nat Med. 2008 Vol. 14(9) pp. 979-84

In a recent publication in Nature Medicine a new transgenic Tet-On Advanced mouse line for Tet-regulated gene expression studies specifically in the kidney is described. Researchers from R.Koesters lab in Heidelberg, Germany used control elements of the mouse Pax8 promoter to direct high levels of expression of Tet-On Advanced (rtTA2s-M2) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice the authors describe a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. In a second transgenic model the expression of transforming growth factor-beta1 is targeted to the kidney, which leads to the successful establishment of an inducible model of renal fibrosis. Finally the authors set up a conditional knockout model using the Pax8- Tet-On Advanced mouse line in combination with a Tet-inducible Cre line LC1. In this combination an inducible deletion of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8- Tet-On Advanced mice as a new powerful tool for conditional modelling of renal diseases in transgenic mice.

Sporadic autonomic dysregulation and death associated with excessive serotonin autoinhibition.

08/28/2008
Audero et al. Science 4 July 2008, Vol. 321. no. 5885, pp. 130 - 133

Sudden infant death syndrome (SIDS) is the leading cause of death in the postneonatal period in children in developed countries. Although the conditions resulting in death are not understood, there is evidence mainly from anatomical studies that point to abnormalities in the brain's serotonin system which makes infants susceptible for SIDS. As such, significant differences in the number of serotonin receptors and serotonergic neurons are detected in the brainstem of SIDS babies which are suspected to cause altered serotonin homeostasis, leading to sudden death.

In a recent publication in Science Cornelius Gross and colleagues from the European Molecular Biology Laboratory (EMBL) in Monterotondo, Italy, report their findings on transgenic mice with deficient serotonin regulation. The genetically modified mice carry a Tet- controlled expression cassette of the serotonin 1A autoreceptor. Mice conditionally overexpressing Htr1a (Htr1aRO) developed normally until day 21 but then died to a high percentage before reaching adulthood. Death could be prevented by treating these mice with Doxycyline, thereby reverting receptor overexpression. Careful radiotelemetric monitoring revealed that bradycardia and hypothermia always preceeded death indicating a general failure to maintain sympathetic tone. Infants that have been monitored during SIDS events displayed similar symptoms. Thus in contrast to serotonin knockout mice which are viable, this novel Tet-controlled disease model mimics critical aspects of this fatal syndrom and may help identify diagnostic markers and prophylactic measures for the prevention of SIDS..

A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types.

07/11/2008
Wernig et al. Nat Biotechnol. 2008 Jul.1 Epub ahead of print

Pluripotency can be induced in somatic cells by ectopic expression of transcription factors Oct4, Sox2, Klf4 and Myc. So far this is done by infecting fibroblasts with high-titer retroviral vectors, which results in reprogramming and leads to the formation of induced pluripotent stem cells (iPS) at a low frequency. In a recent publication members from R.Jaenisch?s lab at MIT have significantly enhanced this process by using genetically modified, homogenous fibroblasts and Tet- regulated expression of reprogramming factors.

In a first step, iPS cells were produced by infecting fibroblasts containing the Tet-On Advanced transactivator with lentiviruses carrying Tet-inducible reprogramming factors and subsequent reprogramming after addition of Doxycycline. Pluripotent stem cells were selected and chimeric mice were generated. In a second step Wernig et al. used fibroblasts from the iPS derived chimeric animals which now contain identical proviral insertions. Reprogramming efficiency was 25-50 fold enhanced compared to previous approaches. This method significantly alleviates factor-based reprogramming and will thus facilitate studies on the molecular events leading to epigenetic reprogramming. Furthermore this approach will allow generation of large numbers of identical cells required for high throughput screens for chemicals replacing the original reprogramming factors.

Site-directed, virus-free, and inducible RNAi in embryonic stem cells.

03/20/2008
Wang, J. et al. 2007 PNAS 104 (52): 20850-20855

Pluripotency can be induced in somatic cells by ectopic expression of transcription factors Oct4, Sox2, Klf4 and Myc. So far this is done by infecting fibroblasts with high-titer retroviral vectors, which results in reprogramming and leads to the formation of induced pluripotent stem cells (iPS) at a low frequency. In a recent publication members from R.Jaenisch?s lab at MIT have significantly enhanced this process by using genetically modified, homogenous fibroblasts and Tet- regulated expression of reprogramming factors.

In a first step, iPS cells were produced by infecting fibroblasts containing the Tet-On Advanced transactivator with lentiviruses carrying Tet-inducible reprogramming factors and subsequent reprogramming after addition of Doxycycline. Pluripotent stem cells were selected and chimeric mice were generated. In a second step Wernig et al. used fibroblasts from the iPS derived chimeric animals which now contain identical proviral insertions. Reprogramming efficiency was 25-50 fold enhanced compared to previous approaches. This method significantly alleviates factor-based reprogramming and will thus facilitate studies on the molecular events leading to epigenetic reprogramming. Furthermore this approach will allow generation of large numbers of identical cells required for high throughput screens for chemicals replacing the original reprogramming factors.

The genetic design of signalling cascades to record receptor activation.

03/20/2008
Barnea, G. et al. 2008 PNAS 105: 64-69

In a recent publication in PNAS, researchers from Sentigen Biosciences in collaboration with R. Axel?s laboratory at Columbia University, established a novel system to monitor protein interactions which was applied to GPCRs, receptor tyrosine kinases and steroid hormone receptors. A fusion protein consisting of a membrane receptor and a Tet-regulated activator tethers Tet-Off to the cell membrane. Sequences of both proteins are separated by a linker containing a protease cleavage site. A second fusion protein links a viral protease to a cellular protein that interacts only with the activated receptor. Upon ligand binding to the receptor the protease is recruited to its cleavage site and will release the Tet-off transcription factor which subsequently will activate reporter gene expression. This so-called ?Tango- assay? converts the transient response of ligand binding to a receptor into a stable and easily quantifiable reporter gene signal thus allowing to record receptor activation without interference from endogenous signaling pathways. The assay was developed for the three different types of receptors mentioned above and further on was shown to allow identification of endogenous ligands for orphan receptors as exemplified for GPR1.

Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver.

29/03/2007
Stanger BZ et al. 2007 Nature 445:886-891

Understanding of the mechanisms and key determinants that control vertebrate organ size is rather limited. In a recent publication in Nature, Stanger et al. elegantly demonstrated that the number of progenitor cells determines final organ size of the pancreas rather than endocrine factors or extrinsic signals. Using transgenic mice expressing diphtheria toxin (DTA) under Tet-control, they selectively destroyed pancreatic progenitor cells at distinct time points during embryogenesis. Newborn mice with progenitor cell ablation during the critical embryonic period showed nearly a complete absence of the pancreas. Furthermore, when the number of progenitor cells was reduced due to DTA expression during a brief period early in development, prenatal embryos reproducibly had significantly smaller pancreata, indicating that the remaining progenitor cells were unable to compensate for the reduced cell number. Performing similar experiments in mice containing Tet regulated DTA expression directed against hepatic progenitor cells demonstrated that, in liver growth, compensation occurs rapidly even after severe reduction of hepatic progenitor cell numbers. Thus, while the number of progenitor cells is a critical determinant of pancreas size, liver size determination relies on extrinsic factors independent from the progenitor cell pool size.

Dissecting self-renewal in stem cells with RNA interference.

09/07/2006
Ivanova N. et al; Nature. 2006 Jun 11

In one of the most recent applications of the Tet-System, scientists at Princeton University identified key regulators of stem cell renewal and differentiation in mouse ES cells. Starting from a large number of down-regulated genes detected in a microarray screening approach on mouse ES cells undergoing retinoic acid-induced differentiation, candidates suspected in the control of stem cell fate were identified. Transduction of murine ES cells with lentiviral vectors encoding constitutively expressed shRNAs directed against each of the suspected regulators was performed to measure ES cell differentiation in a fluorescence-based competition assay. Down regulation of several target messenger RNAs was associated with morphological changes and loss of alkaline phosphatase activity, indicative of a differentiated phenotype. In order to provide functional evidence for involvement of the suspected regulators in self-renewal of ES cells, a genetic complementation strategy (rescue) was devised. For this purpose, the original shRNA lentiviral vectors were modified to allow Tet-induced expression of the shRNA-targeted candidates. Transduction of Tet-On-expressing ES cells with suspected regulators under Tet control revealed that self-renewal became doxycycline-dependent, suggesting specific biological roles as potent regulators of stem cell renewal for seven of the identified genes.

Regulatable Gutless Adenovirus Vectors Sustain Inducible Transgene Expression in the Brain in the Presence of an Immune Response against Adenoviruses.

Xiong W. et al; J Virol. 2006 Jan;80(1):27-37.

In a recent publication in Journal of Virology, Xiong and colleagues evaluated high-capacity adenoviral vectors encoding the improved synthetic Tet-On transactivator (rtTA2S-M2) and tTS the tetracycline-controlled transcriptional silencer for their usefulness as clinical vectors. As expected they showed that in vivo reporter gene expression was high in the presence of Dox and could be downregulated to basal levels after removal of the inducer. Most remarkably regulation of expression also worked in adenovirus-immunized animals proving that Tet-dependent regulation of gene expression is effective in vivo even in the presence of a systemic immune response against the vector.