I Expanding the applicability of the Tet regulatory systems

U. Baron, S. Freundlieb, R. Löw, Ch. Schirra-Müller (Volkswagen-Stiftung, EG, BioRegio)

Though very tight regulation of gene expression may be achieved with the Tet regulatory system - demonstrated 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 suppression 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), tetracycline (Tc) controlled silencers (tTS) were developed that bind to the operator sequences within Ptet (Fig. 1) and shield the promoter from outside activation. Addition 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 retroviruses where single genomes can be delivered to the nucleus. Several vehicles including lentiviral vectors (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 development of tightly controllable gene delivery systems that may become useful in gene therapy. Several collaborations in this area are ongoing.
 

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

Most of the TetR mutants that have advantageously modified the Tet regulatory system resulted from powerful genetic approaches in E.coli. However, the phenotypes observed in the prokaryotic cell do frequently not correlate with those seen in the eukaryotic environment. 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 functional domains can be directly explored e.g. by screening or selecting for activation or silencing of marker functions. First screens revealed a number of interesting TetR mutants of which one - identified in the lab in Erlangen - exhibits a striking reverse phenotype that is superior to the originally described rtTA. Furthermore, we are interested in whether tTA/rtTA mutants can be identified which discriminate between different tetracycline derivatives or which may even be susceptible 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 vivo
S. Berger, M. Hasan, R. Löw, K. Schönig (BioRegio, EG, Volkswagen-Stiftung)

Our transgenic mouse lines that synthesize tTA or rtTA specifically in hepatocytes and in specific areas of the brain, respectively, were used to measure kinetics of induction and shut-off of gene activities in these two compartments of the animal. Using a non-invasive approach that monitores luciferase activity in live animals, repeated cycles of gene activation and deactivation 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, independent 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 bidirectional 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 doxycycline 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. Therefore, 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 collaborations to generate animal models for human diseases. One exciting example is a mouse model for prion disease that is being developed by S. Prusiner and P. Tremblay, UCSF, San Francisco, USA.
 


II   The merozoite surface protein 1 of the human malaria parasite Plasmodium 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 vaccine candidate against infection of P.falciparum, the most lethal one among the human malaria parasites, relies on the subunit concept where individual components of the parasite are utilized to elicit a protective immune response. Our studies focus on a 190 kDa protein which is the major surface protein of the merozoite (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 cellular 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.
 

Synthetic msp-1 genes provide material for immunological, structural and functional studies
C. Epp, C. Fernandez-Becerra, C. Schmid, I. Türbachova, N. Westerfeld (BMBF, DFG, University, Fonds d. Chem. Industrie)

P.falciparum DNA has an exceptionally high AT content 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 proteolytic cleavage sites were reconciled in the design of our synthetic genes, and accordingly expression vehicles 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 concentrate 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-epidemiological surveys
C. Epp, I. Idler, I. Türbachova, S. Weerasuriya (BMBF, DFG, University, Fonds d. Chem. Industrie)

The availability of the various MSP-1 processing products in apparently native conformation has enabled us to develop an ELISA that can be used for serum analysis. In a first approach, we have examined the sera from two trials in which Aotus monkeys were immunized with MSP-1 and challenged with merozoites (collaboration with S. Herrera, Cali, Colombia). Full protection was achieved in 60 % of the animals. Interestingly, protection correlated well with the humoral response against certain areas of MSP-1. Using the new ELISA, we are now re-examining the sera collected 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 erythrocytes
P. Burghaus, I. Türbachova (BMBF, DFG, University, Fonds d. Chem. Industrie)

As the major protein at the surface of merozoites, MSP-1 has been implicated in early association events during the erythrocyte invasion. Following the successful strategy described for the Duffy binding protein, we have exposed full length MSP-1 as well as stepwise truncated versions of the protein on the surface of HeLa cells, fixed, as in the parasite, by a GPI-anchor. Moreover, in collaboration with D. Soldati (ZMBH), we have generated several recombinant Toxoplasma gondii strains that also expose at the surface full size MSP-1, the p42 or the p19 processing 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 respective 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, therefore, will further pursue these studies.


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 conditional 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 candidate MSP-1 from Plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full length protein from E.coli and mammalian cells. Nucl. Acids Res. 27, 1094-1103.

Redfern, C.H., Coward, P., Degtyarev, M.Y., Lee, E.K., Kwa, A., Hennig-hausen, L., Bujard, H., Fishman, G.I. and Conklin, B.R. (1999). In vivo conditional expression and signaling of a specifically designed Gi-coupled 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 Plasmodium 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 susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-kB activation. Nat. Med. 6, 573-577.

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

Mansuy, I. and Bujard, H. (2000) Tetracycline-regulated gene expression in the brain. Curr. Op. Neurobiol. 10, 593-596.

Urlinger, S., Baron, U., Thellmann, M., Hasan, M., Bujard, H. and Hillen, W. (2000). Exploring the sequence space for tetracycline dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97, 7963-7968.
 


Diploma

Epp, Christian (1998):
Analyse der humoralen Immunantwort von Aotus-Affen gegen eine Immunisierung 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 Oberflächenprotein 1 von Merozoiten (MSP-1) des Malariaerregers Plasmodium falciparum: Kontrollierte Synthese des 42kDa-Fragments in Säugerzellen
 

Dissertations

Baron, Udo (1998):
Weiterentwicklung der Methodik der Tetrazyklin-kontrollierten Genexpression zur Analyse der Funktion von Genen in komplexen eukaryotischen Systemen.


Awards

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


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