Research Report 2010 - MDC

have identified an “active” copy of novel endogenousretrovirus in the rat genome, and showed that the elementis active in retrotransposition. The transpositionallyactive copy has an intact env gene, so elementmight be capable of infection.Genome manipulation – Transposon mutagenesisin rat spermatogonial stem cellsLajos Mátés, Ivana GrabundzijaTransposons can be harnessed as vehicles for introducingmutations into genes. Our goal is to establish toolsbased on SB as well as on the piggyBac and Tol2 transposonsystems to manipulate vertebrate genomes(transgenesis, genomic screens) in organisms wherethis technology was not available before. One particularapplication relates to loss-of-function insertionalmutagenesis in rats with the goal of generating knockoutanimals. The genes inactivated by transposoninsertion are “tagged” by the transposable element,which can be used for subsequent cloning of themutated allele. With the goal of knocking out genesimplicated in disease, we carried out a pilot screen inrat spermatogonial stem cells. The project has enormouspotential to develop powerful genomic tools forrat that is the preferred model organism of cardiovascular,as well as toxicology and behavioral studies.Deciphering the genetic background of neuroblastomaand hormone-induced breast cancerSanum BashirThe SB transposon is suitable for somatic mutagenesisand emerged as a new tool in cancer research as analternative to retroviral mutagenesis. Transposonbasedinsertional mutagenesis screens are able to identifiyboth oncogenes and tumor-suppressor genes. Weare engaged in two projects aiming at the discovery ofnovel driver mutations that are associated with neuroblastoma(in mice) and estrogen-induced mammarycancer (in rats). The transposon mutagenesis approachis expected to be a powerful tool to decipher gene regulatorynetworks cooperating in cancer development,progression and metastasis.Transposons as non-viral vectors for genetherapeutic approachesIsmahen Ammar, Csaba Miskey, Katrin Voigt,Jichang Wang, Frank SchulzeDNA-based transposons are natural gene delivery vehicles,and molecular reconstruction of SB represents acornerstone in applying transposition-mediated genedelivery in vertebrate species, including humans. Ourrecently developed 100-fold hyperactive SB systemStable gene transfer and expression of green fluorescent protein (GFP)in differentiating, human hematopoietic stem cell lineages (A-C). Thecells only appear green if the GFP marker gene stably integrated intheir chromosomes by transposition. The hyperactive transposase(SB100X) generates far more green cells than an earlier version of theSB transposase (SB32X).opened new avenues for gene therapeutic approaches,and we are currently developing preclinical animalmodels for gene therapy of Wiskott-Aldrich syndrome,chronic granulomatous disease and Goucher disease.SB transposition occurs into chromosomes in a randommanner, which is clearly undesired for human applicationsdue to potential genotoxic effects associatedwith transposon integration. We succeeded in targetingSB transposition into predetermined chromosomalloci. We employed modular targeting fusion proteins, inwhich the module responsible for target binding canbe a natural DNA-binding protein or domain, or an artificialprotein such as a designer zinc finger. Targetedtransposition could be a powerful method for safetransgene integration in human applications.Selected PublicationsIvics, Z, Li, M.A., Mátés, L, Boeke, JD, Nagy, A, Bradley, A, Izsvák, Z. (2009)Transposon-mediated genome manipulations in vertebrates. NatureMethods 6(6), 415-422.Mátés, L, Chuah, MK, Belay, E, Jerchow, B, Manoj, N, Acosta-Sanchez, A,Grzela, DP, Schmitt, A, Becker, K, Matrai, J, Ma, L, Samara-Kuko, E,Gysemans, C, Pryputniewicz, D, Miskey, C, Fletcher, B, VandenDriessche, T,Ivics, Z., Izsvák, Z. (2009). Molecular evolution of a novel hyperactiveSleeping Beauty transposase enables robust stable gene transfer invertebrates. Nature Genet. 41(6), 753-761.Sinzelle, L, Kapitonov, VV, Grzela, DP, Jursch, T, Jurka, J, Izsvák, Z, Ivics, Z.(2008). Transposition of a Reconstructed Harbinger Element in HumanCells and Functional Homology with Two Human Cellular Genes. Proc.Natl. Acad. Sci. USA, 105(12):4715-20Wang, Y, Liska, F, Gosele, C, Sedová, L, Kren, V, Krenová, D, Ivics, Z, Hubner, N,Izsvák, Z. (2009) A novel active endogenous retrovirus family contributesto genome variability in rat inbred strains. Genome Res. [Epub ahead ofprint] PMID: 19887576Ivics, Z, Katzer, A, Stüwe, EE, Fiedler, D, Knespel, S, Izsvák, Z. (2007). TargetedSleeping Beauty transposition in human cells. Mol. Ther. 15, 1137-1144.Cardiovascular and Metabolic Disease Research 53