I Expanding the Applicability of the Tet Regulatory Systems

U. Baron, S. Freundlieb, R. Löw, Ch. Schirra-Müller

Though very tight regulation of gene expression may be achieved with the Tet regulatory system - dem-onstrated by the successful control of the diphteria toxin gene in transgenic mice most impressively - it is sometimes difficult to initially establish cell lines or transgenic animals where genes encoding potentially toxic products are under Tet control. The main reason for such failures is the transient state of the transferred DNA. During this period when chromatin suppres-sion is missing, background expression from multiple copies of the target gene may be sufficient to kill the cell. We have deviced two approaches that help to overcome this limitaton. By fusing transcriptional silencing domains to the Tet repressor (TetR), tetracy-cline (Tc) controlled silencers (tTS) were developed that bind to the operator sequences within Ptet (Fig. 1)

and shield the promoter from outside activation. Addi-tion of doxycycline (Dox) will dissociate tTS from Ptet and at the same time cause its activation via rtTA. The second approach makes use of gene transfer via ret-roviruses where single genomes can be delivered to the nucleus. Several vehicles including lentiviral vec-tors (collaboration with L. Naldini, Candiolo/Torino, Italy) were composed, which function well. Together, these components should not only abrogate present limitations, they offer themselves also for the develop-ment of tightly controllable gene delivery systems that may become useful in gene therapy. Several collabo-rations in this area are ongoing.

Herrmann Bujard

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Figure 1: The family of Tc-controlled regulatory proteins. TetR (red) was originally fused to a portion of the Herpes simplex virus activator protein VP16 (green) resulting in tTA (Tc controlled transactivator). Replacing the VP16 moiety by multiples of 13 amino acid long minimal activa-tion domains (upper left) has increased the specificity of tTA. A quadruple mutant of TetR exhibiting a reverse phe-notype (violet) was fused to activation domains resulting in rtTA that requires Dox for operator binding. Modifying the DNA recognition specificity of tTA and rtTA (blue) as well as the dimerization surface of tTA (grey) led to tTA and rtTA versions that can be used to control two genes in a mutually exclusive way by Dox. Fusion of TetR with an altered dimerization surface to silencing domains led to Tc-con-trolled transcriptional silencers (tTS) that, in concert with rtTA, establish a repression/ activation system where in the „OFF“ state, i.e. in absence of Dox, Ptet - a minimal pro-moter fused to an array of 7 tet operators - is shielded from outside activation.-

Probing the sequence space of TetR T. Baldinger, U. Baron, M. Hasan, Ch. Schirra-Mül-ler

Most of the TetR mutants that have advantageously modified the Tet regulatory system resulted from pow-erful genetic approaches in E.coli. However, the phe-notypes observed in the prokaryotic cell do frequently not correlate with those seen in the eukaryotic envi-ronment. Therefore, two eukaryotic genetic systems were conceived; one developed by W. Hillen and his group (Universität Erlangen) is based on S.cerevisiae, the other on mammalian cells. With these systems, the sequence space of fusions between TetR and func-tional domains can be directly explored e.g. by screen-ing or selecting for activation or silencing of marker functions. First screens revealed a number of interest-ing TetR mutants of which one - identified in the lab in Erlangen - exhibits a striking reverse phenotype that is superior to the originally described rtTA. Further-more, we are interested in whether tTA/rtTA mutants can be identified which discriminate between differ-ent tetracycline derivatives or which may even be sus-ceptible to non-tetracycline compounds as inducers. If successful, this search could lead to well defined and largely homologous regulatory circuits that would allow to control several genes independently from each other by just using different inducer molecules (collaboration with W. Hillen and B. Berkhout).

Controlling genes in vivoS. Berger, M. Hasan, R. Löw, K. Schönig

Our transgenic mouse lines that synthesize tTA or rtTA specifically in hepatocytes and in specific areas of the brain, respectively, were used to measure kinet-

ics of induction and shut-off of gene activities in these two compartments of the animal. Using a non-inva-sive approach that monitores luciferase activity in live animals, repeated cycles of gene activation and deac-tivation can be followed in individual mice (Fig. 2). These studies have provided not only insights into the time course of switching between active and inactive states of a gene via Dox in live animals but also into the longevity of the expression system. Thus, indepen-dent of whether the luciferase reporter gene was kept in the „ON“ or in the „OFF“ state, regulatory cycles could be re-initiated after 6 months in all individuals tested. As any target gene may be coregulated together with the luciferase gene as indicator via the bidirec-tional Ptet promoters, monitoring luciferase activity in live animal will be indicative for the activity of the gene under study (Fig. 2). In summary, the newly developed rtTA‘s together with some novel doxycy-cline derivatives that are presently being characterized and the induction studies have made us confident that genes of interest can be controlled in the mouse brain tightly and reliably over long periods of time. In this context, it is our aim to control gene functions indirectly by interferring at the level of transcription initiation and/or in post-transcriptional processes, an approach which would leave the endogeneous gene of interest untouched in its genomic setting. There-fore, we explore several experimental options which include padlock RNA‘s, ribozymes and zinc finger proteins. We have chosen the NRI subunit of the NMDA receptor as primary target for this work. Our technology is used in several rather challenging col-laborations to generate animal models for human dis-eases. One exciting example is a mouse model for prion disease that is being developed by S. Prusiner and P. Tremblay, UCSF, San Francisco, USA.

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Synthetic msp-1 genes provide material for immunological, structural and functional stud-iesC. Epp, C. Fernandez-Becerra, C. Schmid, I. Türbach-ova, N. Westerfeld

P.falciparum DNA has an exceptionally high AT con-tent which amounts to 76 % in the coding area of MSP-1. The high AT content has prevented the stable cloning of large genes of this parasite and thus severely hampered their study. We have synthesized the genes of both MSP-1 prototypes (together around 10 000 bp) based on human codon frequencies and are now in a position to produce MSP-1 and derivatives thereof in various heterologous systems. During maturation of merozoites, MSP-1 undergoes proteolytic cleavage resulting in five major fragments that can be isolated as one complex from the merozoite surface. The pro-teolytic cleavage sites were reconciled in the design of our synthetic genes, and accordingly expression vehi-cles encoding the various fragments are available as well. Efficient expression of the intact genes as well as of DNA encoding the processing products has been achieved in E.coli. Full size MSP-1 is also produced and secreted into the medium by bacterial L-forms (collaboration with M. Kujau, IMB, Jena) from where it can be isolated in soluble form. We now concen-trate on devicing a production procedure for MSP-1 that is suitable for „good manufacturing practice“. Our highly purified preparations will also be used for structural studies and for exploring interactions between the individual processed domains as well as between MSP-1 and the erythrocyte surface. A second focus of this project is the integration of msp-1 into viral systems that are suitable as vaccine carriers in humans, such as vaccinia and measles viruses.

A MSP-1 based ELISA for sero-epidemiologi-cal surveysC. Epp, I. Idler, I. Türbachova, S. Weerasuriya

The availability of the various MSP-1 processing prod-ucts in apparently native conformation has enabled us to develop an ELISA that can be used for serum anal-ysis. In a first approach, we have examined the sera from two trials in which Aotus monkeys were immu-nized with MSP-1 and challenged with merozoites (collaboration with S. Herrera, Cali, Colombia). Full protection was achieved in 60 % of the animals. Inter-estingly, protection correlated well with the humoral response against certain areas of MSP-1. Using the new ELISA, we are now re-examining the sera col-lected during our earlier epidemiological studies in West Africa. Our previous analysis has indicated a correlation between antibody titers towards certain regions of MSP-1 and a reduced risk of reinfection by P.falciparum.We hope that our new analysis will reveal more clearly which regions of MSP-1 may be involved in eliciting a protective humoral response. The ELISA may eventually permit the development of a diagnostic tool with predictive properties.

Attempts to identify interactions between MSP-1 and the surface of erythrocytesP. Burghaus, I. Türbachova

As the major protein at the surface of merozoites, MSP-1 has been implicated in early association events during the erythrocyte invasion. Following the suc-cessful strategy described for the Duffy binding pro-tein, we have exposed full length MSP-1 as well as stepwise truncated versions of the protein on the sur-face of HeLa cells, fixed, as in the parasite, by a

II The Merozoite Surface Protein 1 of the Human Malaria Parasite Plas-modium falciparum

Malaria is one of the most widely spread infectious diseases with around 40 % of the world‘s population living in areas at risk. The hope for an effective vac-cine candidate against infection of P.falciparum, the most lethal one among the human malaria parasites, relies on the subunit concept where individual com-ponents of the parasite are utilized to elicit a protec-tive immune response. Our studies focus on a 190 kDa protein which is the major surface protein of the mero-

zoite (MSP-1), one of the parasite‘s blood forms. This protein is believed to play a role during invasion of erythrocytes by the parasite. It is a target of the human immune response at the humoral as well as at the cel-lular level. Such findings suggest that MSP-1 may elicit a protective immune response when used as a vaccine in humans. The aim of our work is to develop an experimental vaccine that is suitable for human trials. Moreover, we are interested in the function of MSP-1 and its structure at the parasite‘s surface.

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Figure 2: Monitoring gene activation and inactivation in live ani-mals. Mice double trans-genic for tTA and the luciferase gene under Ptet control are injected with luciferin and an-esthesized for 5 min. before they are placed into the Hamamatsu photon counting device Argus 20 (left upper part of fig.). Photons emitted by luciferase active in liver (left most animal) and in brain (right animal) are collected as imaged below. The single transgenic animal in the middle serves as con-trol. Feeding Dox to the “liver mouse” abolishes luciferase activity within 5 days. Removal of Dox in the drinking water re-

establishes luciferase activity within 5 days (“brain mouse” as control). The cycle of switching was repeated with the same animals after 3 months with identical results, except that 2 days of exposure to Dox were sufficient to shut off luciferase activ-ity in the liver. The luciferase gene is driven by a bidirectional Ptet which coregulates the Cre recombinase gene. Luciferase activity correlates with Cre activity. Whole body images of the animals are shown.

Mansuy, I. and Bujard, H. (2000) Tetracycline-regu-lated gene expression in the brain. Curr. Op. Neuro-biol., in press.

Urlinger, S., Baron, U., Thellmann, M., Hasan, M., Bujard, H. and Hillen, W. (2000). Exploring the sequence space for tetracycline dependent transcrip-tional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA, 97, 7963-68.

THESES

Diploma

Epp, Christian (1998): Analyse der humoralen Im-munantwort von Aotus-Affen gegen eine Immunisie–rung mit MSP-1 aus Plasmodium falciparum

Schönig, Kai (1998): Konstruktion und Analyse von „integrierten Regulationseinheiten“ zur Tetrazyklin kontrollierten Genexpression in Säugerzellen

Schmid, Christina (1999): Untersuchungen zum Ober-flächenprotein 1 von Merozoiten (MSP-1) des Mala–riaerregers Plasmodium falciparum: Kontrollierte Syn-these des 42kDa-Fragments in Säugerzellen

Dissertation

Baron, Udo (1998): Weiterentwicklung der Methodik der Tetrazyklin-kontrollierten Genexpression zur Ana–lyse der Funktion von Genen in komplexen eukaryo-tischen Systemen.

Awards

Ruperto Carola Preis 1998 der Universität Heidelberg, for outstanding PhD Thesis to Udo Baron

STRUCTURE OF THE GROUPE-mail: bujardh@sun0.urz.uni-heidelberg.de

Group leader Bujard, Hermann, Prof. Dr.

Guest scientist del Portillo, Hernando, Prof. Dr.*

ResearchAssociate Freundlieb, Sabine, Dr.

Postdoctoral Baron, Udo, PhDfellows Fernandez-Becerra, Carmen, PhD* Hasan, Mazahir, PhD* Löw, Rainer, PhD* Weerasuriya, Siromi, PhD*

Ph.D. students Berger, Stefan, Dipl.Chem. Epp, Christian, Dipl.Biol. Idler, Irina, Dipl.Biol.* Kolster, Kathrin, Dipl.Biol.* Schönig, Kai, Dipl.Biol. Türbachova, Ivana, Dipl.Biol. Westerfeld, Nicole, Dipl.Biol.

Diplomastudents Baldinger, Tina* Schmid, Christina* Techn. assistants Rittirsch, Melanie* Schirra-Müller, Christiane Wahedy, Soraya*

* part of the time reported

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GPI-anchor. Moreover, in collaboration with D. Sol-dati (ZMBH), we have generated several recombinant Toxoplasma gondii strains that also expose at the surface full size MSP-1, the p42 or the p19 process-ing product, respectively. In both, the HeLa cell and the T.gondii system, monoclonal antibodies directed towards conformational epitopes of MSP-1 interact efficiently with the protein at the surface of the respec-tive cells. Nevertheless, so far we were neither able to unequivocally demonstrate an interaction between MSP-1 and erythrocytes nor can we definitely rule out such interactions. The failure to reveal the putative affinity may be due to a number of reasons. We, there-fore, will further pursue these studies.

External Funding

During the period reported, our research was sup-ported by grants from the BMBF (Forschungsschwer-punkt Tropenmedizin), the Volkswagen-Stiftung, the Deutsche Forschungsgemeinschaft (SFB 544 “Kon-trolle tropischer Infektionskrankheiten” and Projekt-’Sachbeihilfen’), from the EU, the BioRegion Rhein-Neckar (BMBF - Fa. Knoll AG) and the Fonds der Chemischen Industrie Deutschlands.

PUBLICATIONS

Tremblay, P., Meiner, Z., Galou, M., Heinrich, C., Petromilli, C., Lisse, T., Cayateno, J., Torchia, M., Mobley, W., Bujard, H., DeArmond, S.J. and Prusiner, S.B. (1998). Doxycycline control of prion protein transgene expression modulates prion disease in mice. Proc. Natl. Acad. Sci. USA 95, 12580-12585.

Baron, U., Schnappinger, D., Helbl, V., Gossen, M., Hillen, W. and Bujard, H. (1999). Generation of con-ditional mutants in higher eukaryotes by switching

between the expression of two genes. Proc. Natl. Acad. Sci. USA 96, 1013-1018

Freundlieb, S., Schirra-Müller, C. and Bujard, H. (1999). A tetracycline controlled activation /repression system for mammalian cells. J. Gene Med. 1, 4-12.

Pan, W., Ravot, E., Tolle, R., Frank, R., Mosbach, R., Türbachova, I. and Bujard, H. (1999). Vaccine can-didate MSP-1 from Plasmodium falciparum: a rede-signed 4917 bp polynucleotide enables synthesis and isolation of full length protein from E.coli and mam-malian cells. Nucl. Acids Res. 27, 1094-1103.

Redfern, C.H., Coward, P., Degtyarev, M.Y., Lee, E.K., Kwa, A., Hennighausen, L., Bujard, H., Fishman, G.I. and Conklin, B.R. (1999). In vivo conditional expres-sion and signaling of a specifically designed Gi-cou-pled receptor. Nature Biotech. 17, 165-169.

Burghaus, P.A., Gerold, P., Pan, W., Schwarz, R.T., Lingelbach, K. and Bujard, H. (1999). Analysis of recombinant merozoite surface protein-1 of Plasmo-dium falciparum expressed in mammalian cells. Mol. Biochem. Parasitol. 104, 171-183.

Lavon, I., Goldberg, I., Amit, S., Landsman, L., Jung, S., Tsuberi, B., Barshack, I., Kopolovic, J., Galun, E., Bujard, H. and Ben-Neriah, Y. (2000) High suscepti-bility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-κB activa-tion. Nat. Med. 6, 573-577.

Baron, U. and Bujard, H. (2000) The Tet repressor based system for regulated gene expression in eukary-otic cells: principles and advances. Methods Enzymol. 327, 659-686.

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