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Transposition and Genome Engineering

Head: Dr. Zoltán Ivics

Transposons ("jumping genes") are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. Transposons offer a new model to study DNA recombination in higher organism, as well as host-parasite interaction. Transposons are also natural gene delivery vehicles that are being developed as genetic tools.

We have reconstructed an active transposon from DNA sequence fossils found in fish genomes. Sleeping Beauty not only represents the first DNA-based transposon ever shown to be active in cells of vertebrates, but the first functional gene ever reconstructed from inactive, ancient genetic material, for which an active, naturally occurring copy either does not exist or has not yet been isolated. Technologies based on Sleeping Beauty gene transfer have been revolutionizing genomic manipulations in vertebrate species.

Research Group

Dr. Zoltán Ivics and his research group (Source: PEI)Dr. Zoltán Ivics and his research group

Dr. Zoltán Ivics (Head)
Dr. Csaba Miskey (Scientist)
Dr. Esther Grueso (Scientist)
Dr. Oliver Walisko (Scientist)
Dr. Attila Sebe (Scientist)
Dr. Nina Fuchs (Guest Scientist)
Marta Swierczek (PhD student)
Lisa Wiedemann (PhD student)
Tanja Diem (Technical Assistant)
Franziska Thiels (Technical Assistant)
Heike Krüger (Secretary)

Research Areas

Our laboratory is following the strategy of understanding the mechanism of transposition and its regulation and translate this knowledge to derive transposon-based genetic tools for genome manipulation or for gene therapy. We are currently focusing on the following research areas.

Transposons as non-viral vectors for gene therapeutic approaches

DNA-based transposons are natural gene delivery vehicles (Figure), and molecular reconstruction of the Sleeping Beauty (SB) transposon represents a cornerstone in applying transposition-mediated gene delivery in vertebrate species, including humans. Our recently developed 100-fold hyperactive SB system opened new avenues for gene therapeutic approaches, and we are currently developing preclinical animal models for gene therapy of monogenic diseases.

The Sleeping Beauty transposon system The Sleeping Beauty transposon systemThe Sleeping Beauty transposon system Source: Zoltan Ivics

(a) Components and structure of a two-component gene transfer system based on Sleeping Beauty. A gene of interest (orange box) to be mobilized is cloned between the terminal inverted repeats (IR/DR, black arrows) that contain binding sites for the transposase (white arrows). The transposase gene (purple box) is physically separated from the IR/DRs, and is expressed in cells from a suitable promoter (black arrow). The transposase consists of an N-terminal DNA-binding domain, a nuclear localization signal (NLS) and a catalytic domain characterized by the DDE signature.

(b) Mechanism of Sleeping Beauty transposition. The transposable element carrying a gene of interest (GOI, orange box) is maintained and delivered as part of a DNA vector (blue DNA). The transposase (purple circle) binds to its sites within the transposon inverted repeats (black arrows). Excision takes place in a synaptic complex. Excision separates the transposon from the donor DNA, and the double-strand DNA breaks that are generated during this process are repaired by host factors. The excised element integrates into a TA site in the target DNA (green DNA) that will be duplicated and will be flanking the newly integrated transposon.

Target site selection and its experimental manipulation

SB transposition occurs into chromosomes in a random manner, which is clearly undesired for human applications due to potential genotoxic effects associated with transposon integration. We succeeded in targeting SB transposition into predetermined chromosomal loci. We are currently investigating the use of modular targeting fusion proteins, in which the module responsible for target binding can be a natural DNA-binding protein or domain, or an artificial protein such as a designer zinc finger. Targeted transposition could be a powerful method for safe transgene integration in human applications.

Loss-of-function insertional mutagenesis with transposons

Transposons can be harnessed as vehicles for introducing mutations into genes. Our goal is to establish tools based on SB as well as on the piggyBac and Tol2 transposon systems to manipulate vertebrate genomes (transgenesis, genomic screens) in organisms where this technology was not available before. One particular application is based on loss-of-function insertional mutagenesis in rats with the goal of generating knockout animals. The genes inactivated by transposon insertion are "tagged" by the transposon, which can be used for subsequent cloning of the mutated allele. With the goal of knocking out genes implicated in disease, we carried out a pilot screen in rat spermatogonial stem cells. The project has enormous potential to develop powerful genomic tools for rat that is the preferred model organism of cardiovascular, as well as toxicology and behavioral studies.

Domesticated, transposon-derived cellular genes

The emergence of new genes and functions is of central importance to the evolution of species, but the creation of new genes by recycling of genetic material from selfish transposons is incompletely understood. DNA transposons carry an attractive and elaborate enzymatic machinery as well as DNA components that have been exapted by the host genome via an evolutionary process referred to as molecular “domestication”, by which a transposon-derived coding sequence gives rise to a functional host gene. For example, one particular copy of the transposase gene of the ancient Hsmar1 human transposon has been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. SETMAR retains ist ability to bind to transposon sequences in vitro, and we are currently investigating the cellular function of this gene by integrated genomic and transcriptomic approaches.

Publications

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