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Partial reprogramming restores youthful gene expression through transient suppression of cell identity

Roux A. et al. bioRxiv 2021

Discovery of pluripotency factors by Yamanaka and colleagues several years ago made it possible to generate patient specific stem cells from his/her own somatic cells. However, during this process of cellular reprogramming cells also acquire carcinogenic mutations, limiting the therapeutic potential of iPSCs.

A new strategy seemed to circumvent the risk of oncogenic mutations: transient reprogramming relies on temporary expression of pluripotency factors reversing features of cellular aging without generating iPSCs.  

In a recent publication scientists from Calico Life Science led by J.C. Kimmel analysed the effects of transient reprogramming at the single cell level. They placed the Yamanaka factors under control of the Tet-On 3G system thus activating their expression only for a limited time and analysed the effects by single cell RNA sequencing. They were able to restore „youthful“ gene expression in adipogenic cells and muscle-derived mesenchymal stem cells but this „rejuvenation“ was also accompanied by a temporary loss of cell identity, bringing back the problem of oncogenic mutations.

As the oncogenic potential of the individual Yamanaka factors varies, Kimmel and colleagues analysed the effects of various factor combinations. Again the Tet-On3G system was instrumental to induce the reprogramming gene set. Surprisingly the authors found that no single factor is indispensable as reducing the number of factors only weakened the reprogramming effect. Introduction of only two factors was still effective in rejuvenation while affecting cellular identity much less than the full set of Yamanaka factor. This indicates that rejuvenation can be uncoupled from loss of cell identity and may indicate avenues how reprogramming can be used to restore youthful gene expression without risking carcinogenic mutations.

Kimmel and colleagues tested this hypothesis by transfecting aged myocytes with Msx1,a multipotency factor which in amphibians is involved in limb regeneration. In contrast to pluripotency factors which allow regeneration into almost any cell type, multipotency factors enable differentiation only into a small subset of cell types. Indeed, transient reprogramming with Msx1 successfully restored youthful gene expression in aged myocytes.

The present publication demonstrates that alternative routes to reprogramming strategies independent of the Yamanaka factors for the development of cell therapies may be possible.

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Exquisitely Specifc anti-KRAS Biodegraders Inform on the Cellular Prevalence of Nucleotide-Loaded States

Lim S. et al. 2021 ACS Cent. Sci.

RAS proteins are GTPases cycling between the GTP-bound active form promoting cell growth and differentiation and the inactive GDP-bound form. Mutant RAS proteins like KRAS are known to be involved in many different forms of human cancer yet such tumors are difficult to treat as the search for inhibitors of KRAS function is challenging and thus far has not been successful.  In contrast to high affinity small molecules that inhibit function of target proteins, targeted protein degradation (TPD) relies on binders of disease-causing proteins marking them for proteasomal destruction.

We recently highlighted a publication by A.Partridge and colleagues of Merck & Co. (MSD) on PROTACs, now the same group has published a paper on K-RAS biodegraders. In ACS Central Science they report on the expression of hybrid proteins comprising a high affinity RAS binding domain fused to an ubiquitin E3 ligase. With the help of their anti-GFP biodegrader platform and various GFP-KRAS fusions under Tet-On 3G control, the authors demonstrated that KRAS can be tagged for degradation and subsequently identified a number of E3s that will induce GFP-KRAS degradation. To demonstrate degradation of endogenous KRAS the authors choose various high affinity RAS binding proteins with differents specificities for e.g. nucleotide-loaded state or isoform. Induction of expression resulted in rapid KRAS degradation with kinetics approaching those of small molecule based systems.

This publication highlights the use of biodegraders as molecular tools and indicates their potential for future clinical development.

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ObLiGaRe doxycycline inducible (ODIn) Cas9 systems driving pre-clinical drug discovery, from design to cancer treatment

Lundin A. et al. 2020 bioRxiv

The CRISPR-Cas9 system has become an indispensable tool for gene editing in cells and animal models. However, generation of disease models in mice still is a time-consuming and costly process.

M. Marcesca and his collaborators at AstraZeneca have generated the ObLiGaRe doxycycline inducible (ODIn) Cas9 system for tight and reversible control of Cas9 using the Tet-On 3G system. Their vector comprises both Tet components on a single DNA. ß-globin insulators flanking the TRE3G-Cas9 expression cassette shield the promoter from activation in the absence of doxycycline (Dox) while inverted zink finger nuclease sites targeting Rosa26 or AAV1 sites facilitate transgene integration in mouse or human genomes.

Marcesca et al. first demonstrated functionality of their system by generating stable cell lines of human and mouse origin as well as hiPSCs. These cell lines showed Cas9 activity only in the presence of doxycycline and efficient editing in 20-60% of cells. In further studies they generated the ODInCas9 mouse that displayed the expected ubiquitous expression pattern of the Cas9 transgene after integration into the Rosa26 locus. Cas9 activity could be repeatedly turned on and off and no nuclease expression was detectable after dox withdrawal, demonstrating tightness of control in vivo conferred by the Tet-On 3G transactivator. The ODInCas9 mouse was finally used to model non-small cell lung cancer adenocarcinomas after adenoviral delivery of sg RNAs targeting Kras or the Trp53 tumor suppressor. In the tumors, editing resulting in the correct mutation in Kras or Trp53 occurred at rates between 7 and 28%.

This system will be extremely useful for fast generation of cell lines for preclinical testing and production of mouse models e.g. for target validation or modeling of cancers.

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bioPROTACS as versatile modulators of intracellular therapeutic targets including proliferating cell nuclear antigen (PCNA)

Lim S. et al 2020 Proc. Natl. Acad. Sci. USA 117: 5791-5800

PROTACs are bifunctional molecules that connect a protein of interest (POI) to an E3 ligase thus tagging the protein for degradation via the ubiquitin protease system (UPS). Further development led to the generation of bioPROTACS consisting of a modified E3 ligase where the target binding domain is directly fused to the E3 ligase.

In a recent publication in PNAS A.W. Partridge and colleagues describe a model system to identify and validate active bioPROTACs directed against GFP fusion proteins. A HEK293 cell line comprising stably integrated the Tet-ON 3G transactivator and H2B-GFP forms the basis for their experimental setup. This cell line was transiently transfected with a plasmid containing the bidirectional TRE3G promoter simultaneously controlling expression of a bioPROTAC and an mCherry reporter as a control for transfection and expression. After induction with Doxycycline (Dox) H2B-GFP fusions were degraded via the UPS. Using this system the authors were able to identify and validate different combinations of GFP binders and E3 ligases. 

To test if this approach is useful for tackling potential therapeutic targets the authors generated bioPROTACs against proliferating cell nuclear antigen (PCNA) a target for cancer therapy. Indeed they were able to demonstrate rapid degradation of PCNA after addition of Dox with concomitant cell cycle arrest. The present study clearly demonstrates that bioPROTACs are useful to advance basic research on protein degradation but may also present a potential approach to clinical therapies.

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Five Years of Successful Inducible Transgene Expression following Locoregional Adeno-associated Virus Delivery in Nonhuman Primates with No detectable Immunity

Guilbaud M. et al. 2019 Hum Gene Ther. 30: 802-813

Recombinant Adeno-Associated Virus  (rAAV) is a preferred vector for gene therapy applications  targeting muscle. However, intramuscular injections often provoke immune responses of the host resulting in loss of transgene expression. In a recent publication in Human Gene Therapy, O. Adjali and colleagues presented the results of a study started 5 years ago to evaluate the effect of the locoregional delivery route. At the time, monkeys (Macaca fascicularis) were injected with rAAV conferring Tet-regulated expression of erythropoietin (Epo). The Tet transactivator was constitutively expressed.  Doxycycline (Dox) was administered orally intermittedly (seven times the first year, after 2 years and after 5 years). Over the entire time period Epo secretion was induced after every Dox administration. Moreover, in contrast to previous studies no immune response was detected against the Tet transactivator. This clearly shows that the Tet system can efficiently support long-term expression of a transgene without induction of an immune response.

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Generation of a genetically encodable system for protein synthesis inhibition using Tet-On3G

A genetically encodable cell-type-specific protein synthesis inhibitor

Protein synthesis is required when external or internal stimuli induce remodeling of cellular function. To analyse the role of protein synthesis, chemical inhibitors like antibiotics are generally used which however lack tissue specificity and suffer from off target effects.

In a recent publication in Nature Methods the lab of Erin Schuman, Max Planck Institute for Brain Research, Frankfurt, Germany describes the use of the Tet-On 3G system to generate a genetically encodable protein synthesis inhibitor (gePSI). Specifically they cloned the two catalitically active chains of a ribosome-inactivating protein (RIP) from maize behind the bidirectional PTRE3G-BI promoter and used the Tet-On 3G transactivator under cell-type specific control to obtain spatiotemporal control of protein synthesis in rat hippocampal neurons. Specifity of this system allowed to reversibly shut down protein synthesis in cells without affecting neighboring cells. This gePSI will be instrumental to determine the role of specific cell populations in various brain functions like memory and learning. Broader application e.g. in cancer research to identify and stop uncontrollably proliferating cells is possible as well.   

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Tet-On 3G System instrumental in understanding toxic effect of Cas9 activity in hPSCs

p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells

The CRISPR-Cas9 technology has become an indispensable tool in biological research and is of enormous value for biotechnology and medical applications. While genome engineering via Cas9 is highly efficient in transformed cells several studies have shown that pluripotent stem cells (hPSCs) are less amenable to gene editing via Cas9.

In a recent publication in Nature Medicine, Kaykas and coworkers established a system that allows indel (insertion or deletion) generation with high efficiency in hPSCs. The system is based on stable integration of an all-in one construct comprising Tre3G-controlled Cas9 and the Tet3G transactivator.  Lentiviral delivery of sgRNAs led to a high rate of gene disruption only when Doxycycline was present in the growth medium indicating tight control of the system. However, efficient indel generation was accompanied by a sharp decrease in cell number indicative for Cas9 related toxicity. Further analysis demonstrated that P53 is responsible for the toxic response to DNA damage induced by Cas9. This provides an explanation for the observation that hPSCs have inefficient rates of genome engineering. At the same time these results have important implications for the development of hPSC-based clinical applications: selection for inactivated P53 in hPSC-based therapies increases the risk of tumorigenesis. Experiments with temporarily inactivated P53 could prove whether this will lead to reduced toxicity while increasing efficiency of genome editing via Cas9 in hPSCs.

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Tet-On 3G technology used to generate stable human endothelial cells from umbilical cord blood or adult bone marrow

Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition

Cardiovascular diseases particularly those arising in arterial blood vessels are the leading cause of deaths worldwide. In vitro studies of the biology and pathology of human arterial endothelium are hampered by methods to grow arterial endothelial cells in culture. Commonly cells are isolated from arteries of cadavers but such cells quickly lose their proliferative capacity and thus are not suitable for detailed studies.

By using the Tet-On 3G System to control expression of two transcription factors (MYCN and SOX17) David Vereide and his team at the 'Morgridge Institute for Research', Madison, Wisconsin were able to stably expand arterial endothelial precursors from human CD34+ cells from umbilical cord blood and adult bone marrow.  After shutting down expression of both transcription factors by removal of doxycycline these cells gave rise to stable cells exhibiting endothelial functions. Moreover, the researchers noted that the cells exhibit 2 variants: (i) some retain their properties as arterial endothelial cells (ii) while others undergo endothelial to mesenchymal transition. This transition is an important risk factor for a variety for diseases like arteriosclerosis, fibrotic disorders, and vascular graft occlusion.  Further studies with cells able to undergo the transition may provide insights into the biology of the change and hopefully lead to the discovery of drugs to treat or prevent it.

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Tet-On 3G System instrumental in understanding toxic effect of Cas9 activity in hPSCs

Tet technology allows growth of blood stem cells from adult human or mouse cells

Haematopoietic stem and progenitor cells from human pluripotent stem cells

Conversion of adult endothelium to immunocompetent haematopoietic stem cells

Recently a milestone has been reached in stem cell research: independently two research groups have succeeded in generating hematopoietic stem cells from mature cells.

The group of Rafii at Weill Cornell Medicine worked with adult mouse endothelial cells which they directly reprogrammed into immature HSC by transfecting them with a set of transcription factors under control of the Tet-On3G system. These cells were subsequently grown on cells taken from umbilical cord to provide an environment suitable for HSC maturation before transfer into mice that had previously been irradiated to kill endogenous blood and immune cells. The animals survived as implanted stem cells had regenerated various types of blood and immune cells. As the transcription factors chosen had previously been linked to leukemia, tumour formation after implantation was a concern. However monitoring transplanted mice for 20 weeks did not show any indication of tumour formation and overall mice lived a full life for more than 1.5 years in the lab.

G. Daley's team at Boston Children's Hospital followed a different route: they used human adult cells (endothelium) which they first reprogrammed into iPS cells by following an established procedure. They then identified a set of 7 transcription factors placed under control of the Tet-On Advanced transactivator that could induce these cells to become immature HSCs before transplantation into the bone marrow of adult NSG mice. After several weeks these transplanted progenitor cells had transformed into precursors of all major blood lineages.

The ultimate assay for HSC is their ability to give rise to all blood derived cell lineages after transfer into a host. Cells fulfilling these criteria have been generated by 2 different routes as summarized above using the Tet-On technology for reprogramming . These research studies present exciting perspectives for the stem cell field and for therapeutic strategies to treat acquired or genetic haematological disorders.

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Tet-On Advanced system is instrumental for continuous production of mature red blood cells from an immortal line of adult erythroblasts

An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells

The demand for safe blood is globally high and will continue to increase as people live longer while numbers of blood donations keep decreasing. As an alternative to donated blood scientists are trying to find ways for the generation of "synthetic blood". To date,  use of stem cells from adult peripheral blood, umbilical cord and pluripotent stem cells yielded limited success as only small quantities of red blood cells can be obtained.

A paper published earlier this year in 'Nature Communications' by scientists from University of Bristol, UK describes a different approach: adult bone marrow stem cells were transduced with HPV16 E6/E7 under control of the Tet-On Advanced system thus generating an immortalized erythroid cell line. These cells proliferated continuously after induction with doxycycline (Dox) and were morphologically characterized as pro- to early basophilic erythroblasts. Expression of HPV16 E6 and E7 was shut down efficiently after removal of Dox and the cells differentiated into mature erythroblasts and reticulocytes which in the future may be used in the clinic.

This method provides a way to continuously manufacture red blood cells for clinical use but it will take years before such cells will be available on a large scale. In the short term this new technology will not replace blood donations but may provide specialist treatment for patients with rare blood groups or patients requiring multiple transfusions like those suffering from sickle cell disease or thalassemia.

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Tet-On 3G System used to strengthen safety profile of CAR T cell therapy approaches

In recent years immunotherapy has evolved as a new form of cancer treatment. Adoptive cell therapy with CAR T cells was shown to hold promise for treating B-cell malignancies.

The CAR T cell method relies on infusion of a person's own T-cells that have been genetically altered ex vivo to express a modified chimeric antigen receptor (CAR) directed against specific molecules on the surface of target cells.  A number of ongoing clinical trials for treatment of B-cell malignancies use CAR T-cells directed against CD19.  CAR T cells are activated upon exposure to CD19 and proliferate until all CD19 bearing cells are eliminated regardless of whether these are malignant or normal B cells. Consequently, constitutive expression of the CAR can lead to severe adverse events like B-cell aplasia. To address this problem suicide gene systems relying on HSV thymidine kinase or inducible Caspase-9 have been developed. These systems effectively eliminate most of the CAR T cells after induction of the suicide gene. However, this may lead to disease progression after elimination of the transferred, engineered T-cells. A system that allows regulation of CAR expression in an inducible and reversible manner would be favourable.

Terakura and colleagues used the Tet-On 3G system to control expression of CAR in T-cells directed against CD19. They used the pRetroX-Tet-One 3G vector from TakaraBio to clone CD19CAR behind the PTRE3G promoter. Addition of Doxycycline (Dox) led to activation of the Tet-On3G transactivator and expression of CD19CAR. Using this system the authors were able to show that Tet-CD19 CAR T cells plus Dox were as cytotoxic against CD19 expressing cell lines as conventional CD19 CAR T cell.  However, CAR expression was turned off in the absence of Dox. In a xenografted mouse model tumor growth was suppressed by Dox in mice that had received Tet-CD19 CAR T cells.

This new form of switchable, regulated CAR T cells may offer an option to overcome limitations of CAR T cell-based therapy like B-cell aplasia.

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Tet-regulated CRISPRi confers specific, reversible and efficient gene knockdown in iPSCs and other cell types

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

 CRISPR-Cas is currently the hottest tool to edit genes to study gene function or disease mechanisms. In the original system (CRISPRn) CRISPR associated nuclease Cas9 is used to delete specific parts of the genome while in CRISPRi (CRISPR interference) a nuclease deactivated version of Cas9 (dCas9) inhibits expression of genes.

In a recently published study in 'Cell Stem Cell' researchers from The Gladstone Institute compared tet-inducible versions of CRISPRn with CRISPRi in inducible pluripotent stem cells (iPSC). To generate iPSC cell lines with both systems, they used a single vector containing an advanced version of the Tet-On transactivator driven by the strong constitutive CAG promoter and the third generation tet promoter (PTRE3G) to control expression of either Cas9 (CRISPRn) or dCas9-KRAB-P2A-mCherry comprising dCas9 fused to a KRAB repression domain. By using the pluripotency factor Oct4 as a target they found that in bulk populations >95% of CRISPRi cells showed efficient knockdown compared to only 60-70% of CRIPSRn cells. Regulation of Cas9 was tight as no leaky expression of neither dCas9-KRAB nor Cas9 was detectable in the absence of Doxycycline (Dox). Addition of Dox lead to selective induction of Cas9 without any concomitant signs of cytotoxicity, decrease in proliferation or change in morphology of the cells.

Further analysis confirmed that the tet-regulated version of CRISPRi is the tool of choice to selectively and reversibly knockdown expression of a range of genes not only in iPSCs but also in iPSC-derived cardiac progenitors, cardiomyocytes and T lymphocytes.

Overall tet-regulated CRISPRi holds great promise as it avoids potential problems associated with the original CRISPR-Cas system like in frame insertions or deletions leading to partial loss- or gain of function phenotypes. Tet-regulated CRISPRi is a powerful technology to reversibly repress gene expression in bulk populations and to investigate the function of genes that affect development and disease.

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New Tet-inducible CRISPR-Cas platforms for studies of gene function

CRISPR-Cas is the newest, simplest and most applied technology for genome editing. What makes this technology particularly attractive for eukaryotic applications is a dramatic decrease in time required to generate new models for studies of e.g. gene function.  To avoid off-target effects of CRISPR associated nuclease Cas and to allow its tissue- and time-specific expression, two recent publications describe platforms that combine CRISPR-Cas and TET Systems' proprietary Tet technology.

D.Huangfu and colleagues of Memorial-Sloan-Kettering Cancer Center (Cell Stem Cell 15: 215 (2014)) were the first to efficiently generate human pluripotent stem cells (hPSCs) with Tet-inducible gene knockouts. Their approach will allow scientists to conduct genome editing at different time points e.g. during the differentiation process.

A recent publication by S. Lowe and coworkers (Nature Biotechnology 33:390 (2015)) applied (inducible) iCRISPR-Cas to genome editing in transgenic mice and showed that Tet-controlled Cas causes target gene alterations comparable to those generated in conventional knockout mice in much less time. Their system will be extremely useful to knock out genes for studies of gene function and to introduce specific nucleotide alterations for precise modeling of disease-associated mutations.

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New Tet-inducible CRISPR-Cas platform to study gene function in vivo

Inducible in vivo genome editing with CRISPR-Cas9

Nature Biotechnology 33: 390-394 (2015)

The CRISPR-Cas technology is currently the preferred genome editing tool of molecular biologists. In bacteria, CRISPR together with associated nucleases like Cas9 acts as a defense system against viral infection. What makes this technology particularly attractive for eukaryotic applications is a dramatic decrease in the time required to generate mouse models for studies of e.g. gene function as there is no need for gene targeting by homologous recombination.

In a recent publication in Nature Biotechnology, Scott Lowe and collaborators established a method to generate knockout mouse models in less than 6 months. As a proof of concept they generated deletions and frameshift mutations in the tumor suppressor gene Apc involved in colon carcinogenesis. The authors used the Tet technology, specifically a reverse transactivator (rtTA) and the TRE3G promoter to control expression of Cas9. They also generated a targeting vector that can accommodate multiple U6-sgRNAs allowing to target several genes. After thorough analysis of their iCRISPR platform in ES cells, Lowe and colleagues generated transgenic mice comprising sgRNAs targeting Apc and Trp53 both driving colon tumorigenesis.  Treatment of young mice with Doxicycline (Dox) induced Cas9 expression and led to the expected gene alterations. Loss of Apc and p53 protein expression was confirmed by immune-staining. Isolation of intestinal crypts from such mice and subsequent treatment with Dox for various times resulted in production of undifferentiated spheroids indicative for cell proliferation as a result of Apc inactivation.

While duration of Cas9 expression can be controlled via exposure to Dox, spatial induction is a result of rtTA expression in a given organ. In the present study hydrodynamic transfection was used to express rtTA in the liver of mice containing sgRNA targeting Apc. Histological analysis confirmed nuclear accumulation of ß-catenin in hepatocytes. Expression via a tissue specific promoter as demonstrated in many mouse models would have been an alternative approach to hydrodynamic transfection to control rtTA expression.

This described technology is an efficient and rapid strategy to generate conditional knockout mice using Tet-inducible CRISP-Cas in less than 6 months.

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Inducible mouse models confirm contribution of bipotent breast stem cells to mammary gland development and potentially breast cancer

In situ identification of bipotent stem cells in the mammary gland.

Nature. 506:322-327 (2014)

Mammary epithelium consists of two cell layers that undergo complex changes during different stages of development of the gland during puberty. There is a longstanding debate on the existence and contribution of bipotent MaSCs (mammary stem cells) to the development of the mammary gland.  

In a recent publication in Nature, Visvander and colleagues of 'The Walter and Eliza Hall Institute of Medical Research' in Australia used a novel 3D confocal imaging strategy with unprecedented resolution to follow the fate of the marked cells and to determine the contribution of stem cells versus progenitor cells. They generated mice expressing a Tet-On transactivator -IRES-GFP construct under control of the transcription factor Elf5 promoter which directs expression specifically to the inner luminal layer. Another, analogous line with the Tet-On transactivator under control of the K5 (bovine keratin 5) promoter was used to direct reporter gene expression to the basal cell layer. Crossing these transactivator mice to a Tet-inducible Cre strain and floxed R26RConfetti reporter alleles followed by high-resolution 3D cell-fate mapping allowed tracing of bipotent MaSCs at a clonal level.

Results of these studies provide unequivocal evidence for the existence and targeting of bipotent stem cells to the basal layer of the mouse mammary epithelium and their contribution to all major stages of breast development. Another result of Visvander's studies points to a much longer lifespan of breast stem cells than expected and that they are active throughout the life of the individual. This implies that genetic defects acquired early in life could be retained over many years before potentially contributing to breast cancer development. Thus these findings may be of importance for the development and diagnostics for breast cancer.

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Tet regulated mouse model provides insights into new strategies for treatment of acute lymphoblastic leukemia

Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia

Genes Dev. 2014 Jun 15;28 (12):1337-50

Acute lymphoblastic leukemia (B-ALL) is the most common cancer affecting children. Loss-of-function mutations in genes encoding transcriptional regulators of lymphoid development have been identified in approximately two-thirds of B-ALL cases. Among these PAX5 gene modifications leading to disrupted DNA-binding or impaired transcriptional regulatory potential are most common. In mice, PAX5 drives B-lineage differentiation by activating B-lineage-specific gene expression, while repressing alternative lineage genes.

To study  how PAX5 is involved in the B-cell tumor development R. Dickins and colleagues from the 'Walter and Eliza Hall Institute of Medical Research' in Australia created a RNAi-based transgenic mouse model allowing reversible, Tet-controlled knockdown of endogenous Pax5 expression in vivo. Transgenic mice were generated by targeting the Tet-controlled promoter into the type I collagen (Col1a1) locus, which is ideally suited for tightly controlled inducible expression. The shRNA targeting PAX5 is expressed together with GFP open reading frame in one transcript (TRE-GFP-shPax5) thus allowing to readily monitor RNAi expression. To obtain pan-hematopoietic expression these transgenic mice were crossed with the previously established Vav- tTA line (Kim W. et al. 2007; Takiquchi et al. 2013)

Using this model R. Dickins and colleagues discovered that switching on the expression of PAX5 even in cells that have already become cancerous was sufficient to reverse B-cell leukemia. Moreover, they demonstrate that PAX5 restoration leads to continuous disease remission despite the presence of other oncogenic mutations. PAX5 expression turns on normal B-lineage differentiation, leading to progressive tumor clearance and long-term survival.

Analysis of the effect of PAX5 expression in human cell lines was also made possible by the Tet technology. Three human B-ALL cell lines harboring different PAX5 aberrations were stably transfected with a second generation Tet transactivator  and the most advanced Tet-On 3G promoter tightly controlling expression of PAX5 coupled to GFP. Induction of PAX5 expression by addition of Doxycycline, the most widely used inducer of the Tet systems, triggered changes in expression of cell surface markers associated with B-cell differentiation indicating proliferative exit.  

In summary, the results of Dickens and coworkers demonstrate that it may be possible to develop specific treatments fighting B-ALL which have the same effect as restoring PAX5 function. These may act faster with fewer side-effects compared to current treatments.

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New target site for anti-cancer drug development discovered

The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

Nature 2012 Vol. 492, 285-289

If not protected properly, the ends of our chromosomes are prone to DNA damage, random DNA end fusion, and even unnatural hyperextension by the telomere-replicating enzyme, telomerase. POT1-TPP1, a heterodimeric subcomplex of the multi-protein complex known as shelterin, associates specifically with telomeric DNA to function in chromosome end-protection. Contradictory to its protective role, TPP1 has also been implicated in recruiting telomerase to telomeres and in stimulating telomerase processivity. So far, analysis of TPP1 to further define this regulatory function has been complicated by the loss of telomere end-protection by genetically altered TPP1.

In the present study, J. Nandakumar and colleagues from T. Cech's laboratory of the University of Colorado at Boulder succeeded in identifying seven critical separation-of-function mutants of human TPP1. While retaining their full telomere capping function, these TPP1 mutants are deficient in telomerase binding and fail to stimulate the enzyme's processivity. When mapped on the surface of TPP1 the altered amino acids cluster to reveal a patch rich in glutamate (E) and leucine (L), which was named the TEL patch. To directly address the physiological significance of telomerase regulation by the TEL patch of TPP1, they attempted to reproduce their in vitro results in human cells. They utilized the Tet-On Advanced system to establish stable HeLa cell lines in which endogenous TPP1 is fully replaced by mutant human TPP1. Under the control of the bidirectional Tet-inducible promoter both an shRNA against endogenous TPP1 and an shRNA-resistant version of Flag-tagged TPP1 (WT or mutant) were constructed.  Stable cell clones were then generated by integrating a single copy of these constructs into a predefined genomic locus of the specific HeLa cell line utilizing Flp-mediated cassette exchange. After 96 hours of 200 ng/ml of Doxycycline (Dox) the endogenous TPP1 was stably replaced with uniform levels of either WT or mutant Flag-TPP1, thereby allowing a direct comparison. The Boulder research team was  able to grow and analyze these stable clones over 81 population doublings in the presence of Dox to demonstrate that the TEL patch mutants of TPP1 fail to stimulate the lengthening of telomeres in human cells while normal telomere lengthening, comparable to what occurs in WT TPP1 cells, was measured in the absence of Dox. In further experiments IF and IF-FISH visualized the Dox-induced TPP1 TEL patch mutants at telomeres within the cell nuclei and showed their lack of interaction with telomerase. Given the identification of the TEL patch on human TPP1 the TEL patch may serve as a new target for anti-cancer drugs that could make the ends of chromosomes inaccessible to telomerase without interfering with their natural protection.

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Further progress in cell replacement strategies with implications for strategies to treat brain diseases

Generation of induced neurons via direct conversion in vivo

Proc Natl Acad Sci U S A. 2013 Vol. 110 (17): 7038-7043 (published ahead of print March 25, 2013).

Direct conversion of fibroblasts into functional neurons may be an attractive alternative to the pluripotent stem cell approach for studies of neuronal diseases and for cell therapy. So far it is possible to directly convert fibroblasts into functional neurons (induced neurons (iN) in vitro. Direct in vivo conversion has been shown in the pancreas and the heart, but not yet in the brain.

In the present study, scientists around A.Björklund and M.Parmar at Lund University in Sweden demonstrate that the same set of neural in vitro conversion factors is sufficient to convert fibroblasts and human astrocytes in vivo into neurons. They used lentiviral vectors carrying the 3 neuronal conversion factors Ascl1, Brn2a and Mytl1 (ABM) under Tet-On control to transduce GFP-labeled human fetal lung fibroblasts (HFL1) and a second lentivirus expressing the Tet-On Advanced transactivator that was cotransduced in all conversion experiments. Transduced HFL1 cells were grafted into the brain of adult rats and expression of conversion factors was initiated by addition of Doxycycline in the drinking water. Four weeks later cells coexpressing GFP and a neuronal marker (hNCAM) were detected, indicating fibroblast to neuron conversion. Conversion had taken place only in those animals which had been grafted with ABM-transduced cells and also exposed to Dox. Doxycycline was administered for 6 weeks only but iN could still be detected after 25 weeks which confirms long-term stability of the induced neuronal phenotype in the absence of exogenous gene expression. Similar results were obtained when human astrocytes were used; this is of particular interest as endogenous astrocytes may be a source of neurons in diseased brains and thus in vivo astrocyte to neuron conversion may open up new strategies for brain repair.

The results reported by Björklund and Parmar are of importance for novel treatments particularly for degenerative diseases such as Parkinson`s Disease (PD) where cell replacement via transplantation has already shown promise in animal studies but possibly also for others like Huntington's disease, amyotrophic lateral sclerosis (ALS) or Alzheimer's disease.

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Preservation of cell integrity during desiccation

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

PNAS 2012 Vol.109: 20859-64

Dehydration of living cells usually leads to cell death. Yet a number of organisms have found ways to survive severe droughts.  Accumulation of oligosaccharides like trehalose along with protective proteins is one functional mechanism observed in animals. LEA (Late Embryongeneis Abundant) proteins are supposed to play an important role in this process. They were first discovered in various plants but more recently have been detected in different animal groups, including insects, crustaceans and nematodes.

In a recent publication Steven Hand and collaborators attempted to enhance desiccation tolerance in mammalian cells. They expressed two LEA proteins from brine shrimp (AfrLEA2 and AfrLEA3) in HepG2 cells under control of the Tet-On 3G system. In addition cells were constitutively expressing an exogenous trehalose transporter (TRET1) to allow trehalose uptake. Subsequent analyses showed significant induction and tight control of LEA protein expression depending on the presence or absence of Doxycycline. Upon desiccation and rehydration, cells without LEA proteins or trehalose exhibited 0% membrane integrity compared to 98% of cells expressing either AfrLEA2 or AfrLEA3 and loaded with trehalose. Surprisingly, expression of Tet-controlled AfrLEA3 alone in the absence of trehalose conferred 94% protection.

Overall the results presented by Hand et al. clearly show that LEA proteins can confer increased desiccation tolerance to mammalian cells. In light of the interest in cell therapies, this is an encouraging step towards cell stabilization for storage in the dried state.

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Tet-Transgenic Rodents: a comprehensive, up-to date database

On Nov. 23, 2012 the article "Tet-Transgenic Rodents: a comprehensive, up-to date database" was published online in Transgenic Research. You can get access to the publication using this link or download here.

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First Tet-On® 3G transgenic mouse line published

Transient, Inducible, Placenta-Specific Gene Expression in Mice

Endocrinology 2012 Vol. 153: 5637-44.

The Tet technology, which is broadly applied for tight and fully reversible control of gene expression in higher eukaryotes was transferred into transgenic mice many years ago. Since then, the Tet transactivator and the responsive promoter, constituting components of this binary expression system, have been optimized and in 2010 the 3rd generation of the technology was launched by Clontech. Meanwhile more than 500 mouse lines expressing components of the Tet technology are available through academic research laboratories or repositories around the world and a compilation of published mouse lines is available on TET Systems' website.

Nihar Nayak and colleagues from Stanford University are studying placental function and pregnancy disorders. They recently published a paper on the generation of the first transgenic mouse line using the newest generation Tet-On 3G. Using a novel, integrase-based, site specific approach they first generated a transgenic mouse line which ubiquitously expresses the Tet-On 3G transactivator under control of the CMV early enhancer/chicken β actin promoter.  Blastocysts from such animals were infected with a lentivirus carrying a PTRE3G promoter -luciferase reporter construct and a constitutively active GFP expression unit for identification of infected cells. By culturing the infected blastocysts the authors were able to measure dose-response curves and kinetics of gene expression after induction by doxycycline using bioluminescence imaging. The signal intensity obtained by the light emitting blastocysts was strongly dose dependent and could be detected as early as 15 min after doxycycline was added to the culture medium. The maximal gene induction was found already after one hour leading to a 1000 fold gene increase. Removal of doxycycline from the culture medium was accompanied by loss of gene expression over the course of 8 h, demonstrating the superior and reversible properties of the 3rd generation Tet components in vivo.

After transferring the blastocytes into pseudo-pregnant foster mothers, fetuses developed normally and in vivo imaging revealed uniform and high level dox-induced gene expression in the transgenic placentas. Most impressively, the authors were able to control reporter gene expression in the same mice at different stages of pregnancy.

This manuscript describes the first transgenic application of the Tet-On 3G technology and demonstrates its improved utility. This model will allow Dr. Nayak and colleagues to further understand placental development and function and the transgenic Tet-On 3G transactivator mouse line also will be very useful for studies by other scientists.

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New approach to fight obesity

Programming human pluripotent stem cells into white and brown adipocytes

Nature Cell Biology Vol. 14: 209-221

The number of overweight people is increasing worldwide and obesity has doubled within the last 30 years. Concomitantly metabolic diseases like diabetes and heart disease are on the rise and adipocyte dysfunction is implied to contribute to disease progression.

Adipogenesis is a natural process leading to generation of white and brown adipocytes. While white fat cells are specialized on energy storage, brown adipose tissue is involved in the maintenance of body temperature. As the number of brown adipocytes decreases with age and is greatly reduced in obese individuals there is increasing interest in better understanding adipogenesis and to explore possibilities to treat obesity by converting white to brown fat cells. So far, studies on adipocyte biology are hampered by the limited lifespan of cultured cells obtained from adipose tissue of patients. Furthermore, differentiation of mesenchymal stromal cells (MSC) and adipose-derived stromal vascular cells (ADSVC) into adipocytes suffers from limited proliferative and differentiation capacity.

Recently Cowan and colleagues published in Nature Cell Biology a method to efficiently differentiate human pluripotent stem cells (hiPSC) into white and brown adipocytes. To that end they cultured hPSCs under conditions favoring generation of mesenchymal progenitor cells (MPCs). Induction of adipogenesis in response to known inducers like insulin, rosiglitazone and dexamethasone was inefficient leading to only a low percentage of white adipocytes. In contrast, transduction of MPCs with 2 lentiviruses expressing the Tet-On regulator and Peroxisome Proliferator-Activated Receptor-γ(PPARG2,(a key regulator of white adipocyte development) under Tet control increased the number of cells showing the morphological characteristics and marker protein expression pattern of white adipocytes in almost 90% of the population.

Similarly, transducing MPCs with lentiviruses expressing various tet-controlled factors favoring brown adipocyte differentiation  leads to cells with multilocular fat vacuoles and high levels of uncoupling protein 1(UCP1), both characteristic for brown adipocytes.  Extensive further characterization of both cell types confirmed the type of differentiated adipocyte.

This elegant application of the Tet technology has allowed the establishment of an efficient method for the generation of white and brown fat cells from hiPSCs as prerequisite to establishing assays to screen molecules that may convert white to healthy brown cell types.

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New insights into tumorigenesis of breast cancer expressing mutant p53

Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway

Cell 2012 Vol. 148(1-2): 244-258

There is much evidence that wild-type p53 acts as a tumor suppressor, while in its mutant form it promotes tumorigenesis in many different types of cancer. In a multi-institutional study lead by Dr. Carol Prives of Columbia University a 3D cell culture model was used to study cellular changes of MDA-derived cells. During breast carcinogenesis mammary tissue architecture is disrupted and the present study used this model to delineate the phenotypic effects of mutant p53 in breast cancer. Utilizing doxycycline regulated shRNA technology developed at CSH directed against mutant p53 the authors could demonstrate that depletion of mutant p53 is sufficient to revert breast cancer cells to a more "œnon-malignant" morphology. The authors could show that dose-dependent progressive loss of malignant characteristics correlated with decreasing levels of mutant p53. Genome wide expression profiling of breast cancer cell lines indicated that a number of key enzymes of the mevalonate pathway were down-regulated when mutant p53 was depleted, pointing to a functional regulation of mutant p53 in this pathway. To investigate the potential therapeutic relevance of their findings, the authors analyzed expression data of mutant p53 and genes of the mevalonate pathway in samples of human breast cancer patients and noticed up-regulation of several members of the pathway in human tumors expressing mutant p53.  Furthermore, in cell culture experiments 'Fatostatin', a cholesterol lowering molecule, prevented mutant p53 from binding to the promoter of the HMG-CoA reductase promoter and also changed 3D morphology of two breast cancer cell lines. Thus, in the future the mevalonate pathway may serve as a therapeutic target for breast cancers bearing mutant p53.  

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New animal models for disease studies

Two recently published papers describe use of the Tet-On System in the generation of transgenic zebrafish and transgenic dogs that will be useful to model various human diseases.

Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish

J.Hepatol. 2011 Aug 31. [Epub ahead of print]

For many years zebrafish has been an important model organism in developmental biology. Moreover, as comparison of the human and zebrafish genome sequence shows conservation of cell-cycle genes, tumor suppressor and oncogenes, zebrafish have gained increasing popularity to model different human diseases including cancer. Characteristics like external development, optical clarity and easy maintenance at relatively low cost compared to other laboratory animals, make zebrafish attractive for chemical screenings. Finally their high rate of reproduction and the fact that zebrafish embryos are permeable to various small molecules make them attractive even for high-throughput experiments.

In their publication Gong and collaborators generated transgenic zebrafish expressing xmrk, a fish oncogene encoding a hyperactive version of epidermal growth factor homolog under control of the Tet-On Advanced System. Use of the zebrafish fabp10 promoter directed expression of the transactivator specifically to the liver as verified by whole mount in situ hybridization. Subsequent analysis revealed that all fish, independent of age and sex, developed hepatocellular carcinoma as long as they were kept in the presence of doxycycline. Removal of doxycycline from the water led to tumor regression to the point where all animals showed liver morphology and histology indistinguishable from control. Furthermore Erk and Stat-5 which are downstream components of the EGFR pathway, showed up-regulation after induction of xmrk and could be targeted by their specific inhibitors demonstrating the potential of this transgenic model for screening and testing of anti-cancer drugs.


Generation of transgenic dogs that conditionally express green fluorescent protein

Genesis 49(6), 2011 [Epub ahead of print]

In 2009 the group of Byeong Chun Lee at the Department of Theriogenology and Biotechnology at Seoul National University generated the first transgenic dogs by somatic cell nuclear transfer, expressing red fluorescent protein under control of a constitutively active promoter. Now the same group reports the second generation of transgenic dogs with conditional, Tet-regulated expression of eGFP.

Canine fetal fibroblasts were infected with a retroviral vector harbouring both, the Tet-On Advanced transactivator controlled by the phosphoglycerate kinase promoter (PGK) as well as the TRE-tight controlled eGFP cassette. In the established fetal Tet-On cells, eGFP could be tightly regulated with >40 fold induction after addition of doxycycline (Dox) to the culture medium. Somatic cell nuclear transfer was applied to generate reconstructed embryos, which were used to generate the transgenic dog line. Treating these dogs with Dox for two weeks, EGFP fluorescence could be observed in the whole body, with no adverse clinical signs. In addition the Tet-On eGFP dog did not shown any "leaky" expression.

As dogs (Canis familiaris) share many common diseases with humans including diabetes and cancer this study opens a new avenue for the development of human disease models.

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Key player in triple negative breast cancer (TNBC) identified

Activation of Multiple Proto-oncogenic tyrosine Kinases in Breast Cancer via Loss of the PTPN12 Phosphatase

Cell 2011 Vol.144: 703-718

On our website we recently "highlighted" two publications on viral toolkits for Tet-inducible RNA interference. An article by Westbrook and collaborators published last month in "Cell" now describes a first successful application of these inducible vectors in uncovering the role of PTPN12 in triple negative breast cancer underscoring the importance of the Tet technology in elucidating the role of key players in complex signaling networks.

Currently breast cancer is classified into types expressing ER/PR or those overexpressing HER2. Medical treatment for these types is available but about 15-20% of breast cancers, called "triple-negative" breast cancers (TNBC) are characterized by the lack of expression of either of these cancer specific proteins. Our lack of understanding of the molecular principles behind this form of breast cancer is reflected in a poor prognosis for patients suffering from this aggressive disease.

Westbrook and colleagues focus on tumor suppressor proteins and identify the tyrosine phosphatase PTPN12 as an important player in TNBC.  Starting with a genetic screen for anchorage independent growth of human mammary epithelial cells (HMEC) they could identify PTPN12 as a top candidate. In their subsequent characterization, the Tet technology was instrumental in proving that PTPN12 is involved in oncogenic transformation and acts as a growth suppressor by antagonizing key receptor kinase pathways. As loss of PTPN12 is a common feature of a large fraction of TNBCs the findings by Westbrook and colleagues support the notion that PTPN12 is a possible therapeutic target for this type of currently intractable form of cancer.

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New viral toolkits for robust, Tet-inducible shRNA expression

Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi

Nature Biotechnology 2011 Vol. 29 (1): 79-83

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

PNAS 2011 108 (9):3665-3670

RNA interference has been instrumental to carry out loss-of-function studies in mammals and has provided new insights into basic cellular mechanisms and the origin of human diseases. Meanwhile RNAi-based approaches are widely applied not only to study gene function but also e.g. in high-throughput screens to identify protein coding or microRNA genes encoding putative drug targets.

Achieving high-level shRNA expression is a major challenge in achieving gene silencing via RNA interference. This is of particular importance for genes involved in cell survival and proliferation where cells with inefficient shRNA expression have a selective advantage and thus will out-compete those with high RNAi expression.  In this respect the use of tetracycline controlled shRNA expression systems is extremely useful as expression can be triggered transiently after disease manifestation, effectively modelling therapeutic intervention.

Two recent publications by G.Hannon, S.Lowe and colleagues and by Th. Westbrook, St. Elledge and their coworkers describe new sets of viral vectors that allow identification, tracking and isolation of cells that productively express Tet-inducible shRNA. To avoid silencing and to ensure robust Tet-inducible shRNA expression, both teams follow similar strategies: a single viral vector carries a Tet-On transactivator and the Tet-inducible shRNA while direct linking of fluorescent reporters to the shRNA allows easy monitoring of conditional shRNA expression.

By indirect fluorescent tracking of shRNA expression these new vectors allow the isolation of shRNAs with anticancer potential in complex cellular and even in animal screening systems which would have been missed otherwise as shown for the Rpa3 gene in the Nature Biotechnology article.

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New approach to cure diabetes

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.

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Turning skin cells into neuronal cells while bypassing the pluripotent, embryonic state

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.

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Tet Technology applied to study the mouse transcriptome

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.

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Tetracyline inducible sterility prevents escape of transgenic animals into the wild

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.

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Use of Flp-Mediated Cassette Exchange for reproducible Tet-inducible expression at specific loci in HeLa cells

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.

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New, Virus-free Methods to Generate iPS Cells

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.

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Metastasis: how does it really work?

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.

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New Mouse Model for Kidney Diseases

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

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.


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Sporadic autonomic dysregulation

Sporadic autonomic dysregulation and death associated with excessive serotonin autoinhibition.

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 preceded 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..

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A drug-inducible transgenic system

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

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.

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Site-directed, virus-free, and inducible RNAi

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

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.

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The genetic design of signalling cascades

The genetic design of signalling cascades to record receptor activation.

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.

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Organ size is limited by the number of embryonic progenitor cells

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

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.

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Dissecting self-renewal

Dissecting self-renewal in stem cells with RNA interference.

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.

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HP-Regulatable Gutless Adenovirus

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.

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