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European Biopharmaceutical Review

Tet Technology and Drug Discovery

Manfred Gossen of BCRT, Berlin brings expert insight into Tet gene regulation and its role in biomedical research

The coordinated expression of genomic information is an essential prerequisite for all living organisms, ranging from bacteria to higher vertebrates. Disruption of the correct gene expression patterns often has severe consequences, such as developmental aberrations, pathological states or death. Conversely, it is hard to imagine today’s biomedical research without technologies permitting experimental control of the expression of endogenous or trans-genes.

Several methods of manipulation based on endogenous eukaryotic systems have been developed, which rely on inducing agents such as heavy metals, steroid hormones, insect hormones or rapamycin. However, these systems are often ‘leaky’, have unwanted side effects and, in general, are limited to cellular systems.

The more successful systems for regulation of eukaryotic gene expression have been derived from unicellular organisms. Site specific recombinases like Cre and Flp have been used successfully for conditional knockout approaches in transgenic mice. However, recombinases delete genetic material and represent an irreversible switch. Truly conditional systems should allow for repeated switching between the on and off states and precise adjustment of gene expression. Only tetracyclineregulated technologies fulfil this requirement. They have found widespread application, working in a broad spectrum of biological systems ranging from cultured cells of different origins to transgenic animals.

Based on the long history of successful applications in highly complex experimental settings, it should be anticipated that Tet technology will also make important contributions in areas where its potential has not yet been systematically exploited. Examples include future clinical applications of gene and cell therapy, where the continuous transgene expression is not desirable, and the use of this reliable inducible expression system in cell-based highthroughput screening assays or for cGMP biologics production. Therefore, this article encourages the evaluation and use of the Tet system for such underused applications by providing the underlying principles of this gene switch and highlighting some of the most visible success stories of this technology.

TETRACYCLINE RESISTANCE IN E COLI

The main functional elements of the Tet system are derived from the Escherichia coli tetracycline resistance operon. The Tet repressor (TetR) is a negative regulator of the tetracycline resistance gene and represses gene expression by binding to operator sequences (tetO) in the promoter. In the presence of tetracycline antibiotics (Tcs), this repression is alleviated and the antibiotic is eliminated from the bacterium. Under selective pressure, TetR has evolved to sense minute, non-toxic levels of Tcs, which results in the complete loss of TetR’s DNA-binding activity by an allosteric mechanism, giving way to expression of the resistance gene. The extremely high specificity of the TetR/tetO interaction, the capacity of TetR to sense very low levels of Tcs, and the dramatic drop of DNA binding activity of TetR below detectable levels whenever it is bound to Tcs make the tet operon particularly useful for adaptation to mammalian cells and organisms.

TET-REGULATED GENE EXPRESSION SYSTEMS IN EUKARYOTIC CELLS

Eukaryotic Tet gene regulation systems are comprised of two elements to control transgene expression: a transcriptional activator protein and a responsive promoter. Eukaryotic adaptation has been successful because the highly evolved DNA-binding and the Tc-binding protein domains remained unaltered in the Tet transactivator fusion proteins. The TetR moiety provides specific DNA-binding and Tc-sensing, whereas the addition of the Herpes simplex VP 16 transactivation domain converted the Tet repressor into a eukaryotic Tet transactivator. Two transactivator types have been developed; they differ by only a few amino acids but offer fundamentally different tetO binding properties:

  • tTA (Tc-controlled transactivator) binds to tetO only in the absence of tetracyclines (=Tet-Off)
  • rtTA (reverse tTA) requires Tc-derivatives such as Dox to bind tetO, allowing gene expression only in the presence of Dox (=Tet-On).

The second component of the Tet expression system is a minimal promoter fused to heptamerised Tet operator sequences (tetO). Binding of the operators by either type of Tet transactivator results in strong expression of the linked gene of interest. The original system was published in 1992 and has undergone numerous improvements in both components (1). The most recent version, Tet-3G, consists of a transactivator with significantly enhanced sensitivity towards the effector substance (Dox) and a further optimised Tet-promoter.

APPLICATIONS OF TET-REGULATED EXPRESSION SYSTEMS

More than 7,200 scientific publications describe the use of the Tet gene switch in various applications, making it one of the most widely used molecular biology technologies. The Tet system has been used in many eukaryotic cell systems to express gene products which interfere with cellular processes or are outright cytotoxic, such as Diptheria toxin A (DTA). Tet switches also play an increasing role in RNA-based knock-down approaches for target validation in isogenically matched cell systems. Through its adaptation to all major model organisms like yeast, Dictyostelium, C elegans, Drosophila, Xenopus, Zebrafish, rodents and non-human primates, Tet technology has made a major contribution to the engineering of transgenic animals for developmental studies and as disease models.

TET TRANSGENIC (TX) ANIMALS

Tet systems offer the possibility to induce target gene expression at any time during embryonal development and to revert pathological conditions at will in embryos and adult animals. These truly conditional models have already changed our understanding of several diseases, including tumour development. Almost 100 tTA/rtTA and more than 200 Tet responsive Tx mouse lines containing target genes under control of a Tet-regulated promoter have been published and many more lines are currently characterised. Many established Tet Tx lines are available from The Jackson Laboratory, the European Mouse Mutant Archive (EMMA), and RIKEN BRC. Today, it is easy to cross-breed newly generated Tet Tx mouse strains with already existing lines in order to generate new interesting animal and disease models.

TET MOUSE MODELS FOR HUMAN DISEASES

Transgenic Tet animals are widely used in basic research and to model and study human diseases. Tet Tx animals allow the quantitative control of individual gene expression in a temporal and cell type-specific manner which may mimic respective human diseases more faithfully. Examples for existing mouse models of human diseases include Type 1 diabetes, asthma, Alzheimer’s disease, B-cell leukaemia and Huntington disease. Interestingly, pathological states could be abrogated or even reversed in some models by repressing the disease-causing gene product demonstrating the power of the Tet gene switch.

NEUROLOGICAL MODELS

The Kandel group generated mouse lines expressing either tTA or rtTA under the control of the αCamKII promoter, which restricts tTA/rtTA expression to defined regions of the forebrain. Tet-controlled expression of dominant negative, αCamKII or calcineurin versions allowed for the study of synaptic plasticity in double Tx animals. They showed that animals could not master a spatial learning task when the dominant negative version of the αCamKII gene was expressed. Animals which had first acquired the spatial learning programme lost their memory after the dominant-negative proteins were induced, but could regain their spatial memory when the dominant proteins were turned off (2). These findings suggest that αCamKII is required for learning and memory formation as well as for memory information retrieval.

DIABETES

The power and ‘tightness’ of the Tet gene switch has been documented by a study in which the DTA gene was placed under Tet control. Considering that a few DTA molecules are sufficient to kill cells, it is an amazing achievement that such Tx animals could have been established. When these animals were crossed with Tx animals expressing a Tet transactivator in a tissue-specific manner, double Tx mouse lines were established and thrived as long as DTA expression was suppressed. Induction of DTA production and selective ablation of the targeted cells or tissues was initially established for cardiomyocytes by the Fishman group (3). This strategy was later adapted by the Melton group to study pancreatic and hepatic development (4). Insulinproducing pancreatic beta-cells were either destroyed (type-1) or dysfunctional (type-2), and the resulting unregulated elevated blood glucose levels eventually led to diabetes.

New treatment modalities like generation of beta-cells from stem cells, expansion of existing beta-cells, or conversion of other cells into functional beta-cells are currently under investigation. Thorel et al used the DTA ablation technology to destroy 99 per cent of the beta-cells in adult mice (5). However, functional beta-cells slowly repopulated pancreatic islets in the following months. Using cell tracing studies, they could exclude the possibility that the cell increase resulted from beta-cell proliferation. In a second series of tracing experiments, it was shown that the regenerated beta-cells were actually derived from glucagon-producing alphacells. Apparently, functional beta-cells can be replaced through trans-differentiation of alpha-cells. This knowledge may lead to new diabetes treatment modalities.

CANCER

The possibility to shut down oncogene expression at will enabled the establishment of models to study tumour regression in transgenic animals – an exciting experimental approach in cancer research. Tumour models that overexpress oncogenes such as Myc, H-ras, K-ras, ErbB2, Bcr-Abl1 or SV40 TAg have been developed. An important and surprising conclusion from many of these studies was that oncogene overexpression was required for tumour initiation and maintenance. This was first shown for Myc in the haematopoietic system (6). Inactivation of the initiating oncogene led to complete regression of 90 per cent of the tumours. These experimental studies suggested that targeting a single oncogene could result in an effective tumour therapy. As hypothesised, brief Myc inactivation led to sustained osteogenic sarcoma regression and differentiation of osteogenic sarcoma cells into mature osteocytes. Surprisingly, Myc reactivation failed to restore malignancy, but rather induced apoptosis (7). These and similar results led to the ‘oncogene addiction’ hypothesis. However, only 30 per cent of Myc mammary gland tumours regress upon oncogene inactivation, whereas 60 per cent have acquired a Mycindependent secondary pathway via K-ras mutations (8). Therefore, it is likely that targeting of Myc and K-ras would result in the regression of many known tumours.

The Tet gene switch was also instrumental in the study of metastatic mechanisms (9). These were considered late-stage events in cancer progression, resulting from an accumulation of mutations that ‘enable’ the conversion to full malignancy, including colonisation. However, the Varmus group established a tri-Tx Tet mouse model, in which mammary specific rtTA enables tet control over Myc and activated K-ras oncogene expression. These tri-Tx mice were tumour-free until they were exposed to Dox, which resulted in rapid tumour development. Untransformed cells isolated from these rtTA-MYC/KrasD12 transgenic donor mice never exposed to Dox were injected into the bloodstream of recipient mice, where tumours developed only after Dox exposure. Monitoring oncogene activation at different times after cell transfer revealed that non-transformed cells can reside at ectopic sites for prolonged periods. During this window of time, mutations can accumulate, which results in a fully cancerous state – an important finding for the diagnosis and treatment of human cancer.

STEM CELLS

Stem cells are considered a potential treatment alternative for various diseases, but there is intense discussion concerning the source of such therapeutic materials for regenerative medicine. Adult cells have limited proliferation potential, while the use of foetal or embryonic material raises ethical issues. The observation that fibroblasts (somatic cells) can be dedifferentiated into so-called ‘induced pluripotent stem cells’ (iPS cells) offers an interesting alternative (10). This pluripotency can be induced in somatic cells by expression of a set of transcription factors (Oct4, Sox2, Klf4, Myc). However, this process is tedious and ineffective.

The Jaenisch group has improved this process by using genetically modified, homogenous fibroblasts and Tet-regulated expression of reprogramming factors (11). In a first step, fibroblasts expressing the Tet-On Advanced transactivator were infected with lentiviruses encoding Tet-inducible reprogramming factors. IPS cells emerging after Dox-induced reprogramming were selected and, after turning off these factors, chimeric mice were generated. Fibroblasts from iPS-derived chimeric animals containing identical proviral insertions were used, which increased reprogramming efficiency equal or less than 50 times compared to previous approaches. This de-differentiation method will facilitate studies on molecular events leading to epigenetic reprogramming, and will allow the generation of large cell numbers for high-throughput screens for chemicals to replace the original reprogramming factors. These findings further the development of therapeutic iPS cell production without genetic manipulation.

CONCLUSION

Since 1992, the Tet system has been refined, adapted to a number of biological systems, and applied to new and emerging technologies. This brief review focused on transgenic models, which are already tremendously useful tools for developing new treatment regimens, and on stem cell engineering, where the Tet gene switch has added unparalleled opportunities to adjust transgene expression at will. In both areas, the Tet technology facilitates the translation of scientific advancement into medical progress. Equally important, the vast innovative potential of the existing and future users of Tet technology is likely to result in novel applications not yet anticipated, which will further pharmacological and biomedical research.

References

  1. Gossen M and Bujard H, Tight control of gene expression in mammalian cells by tetracycline-responsive promoters, Proc Natl Acad Sci USA 89: pp5,547-5,551, 1992
  2. Mayford M, Bach M E, Huang YY, Wang L, Hawkins RD and Kandel ER, Control of memory formation through regulated expression of a CaMKII transgene, Science 274: pp1,678-1,683, 1996
  3. Lee P, Morley G, Huang Q, Fischer A, Seiler S, Horner JW, Factor S, Vaidya D, Jaife J and Fishman GI, Conditional lineage ablation to model human diseases, Proc Natl Acad Sci USA 95: pp11,371-11,376, 1998
  4. Stanger BZ, Tanaka AJ and Melton DA, Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver, Nature 445: pp886-891, 2007
  5. Thorel F, Népote V, Avril I, Kohno K, Desgraz R, Chera S and Herrera PL, Conversion of adult pancreatic alpha-cells to betacells after extreme beta-cell loss, Nature 464: pp1,149-1,154, 2010
  6. Felsher DW and Bishop JM, Reversible tumourigenesis by MYC in hematopoietic lineages, Mol Cell 4: pp199-207, 1999
  7. Jain M, Arvanitis C, Chu K, Dewey W, Leonhardt E, Trinh M, Sundberg CD, Bishop JM and Felsher DW, Sustained loss of a neoplastic phenotype by brief inactivation of MYC, Science 297: pp102-104, 2002
  8. D’Cruz CM, Gunther EJ, Boxer RB, Hartman JL, Sintasath L, Moody SE, Cox JD, Ha SI, Belka GK, Golant A, Cardiff RD and Chodosh LA, c-MYC induces mammary tumourigenesis by means of a preferred pathway involving spontaneous Kras2 mutations, Nat Med 7: pp235-239, 2001
  9. Podsypanina K, Du YC, Jechlinger M, Beverly LJ, Hambardzumyan D and Varmus H, Seeding and propagation of untransformed mouse mammary cells in the lung, Science 321: pp1,841-1,188, 2008
  10. Takahashi K and Yamanaka S, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell 126: pp663-676, 2006
  11. Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, Staerk J, Markoulaki S and Jaenisch R, A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types, Nat Biotechnol 26: pp916-924, 2008

 


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Manfred Gossen is Group Leader (Genetic Engineering) at the Berlin-Brandenburg Center for Regenerative Therapies. Manfred is a molecular biologist by training and holds a PhD from Heidelberg University, Germany. He is co-inventor of the Tet-regulated gene expression systems (36 patents) and has authored 39 scientific publications.
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