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Gene Manipulation

Gene Manipulation

Cutting-edge plasmid DNA cloning and gene transfer technologies for next-generation RNA therapeutics.

Gene Manipulation

The Department of Gene Manipulation consists of the Cloning and Gene Transfer Units.

The Cloning Unit provides cutting-edge plasmid DNA cloning services that enable deeper insights into cellular processes at the molecular level and help lay the foundation for the development of new therapeutics. In the Gene Transfer Unit, we apply different approaches to manipulate or transfer genes into host cells. Here, we use retroviral particles to deliver genes for permanent expression, CRISPR-based approaches for gene manipulation and trans-amplifying RNA for therapeutic RNA expression at low doses.

  • Maximilian Mustermann

    Department Director

  • Max Mustermann

    Deputy Director

Our vision

Deepening our understanding of cellular processes and advancing next-generation RNA therapeutics through molecular cloning expertise, genome manipulation and mechanistic research.

Cloning Unit

  • Maximilian Mustermann

    Director Functional Unit

  • Maximilian Mustermann

    Deputy Director Functional Unit

Our activities and competences

Our methods and platforms

Polymerase chain and cloning reactions

Polymerase chain reactions (PCRs) are used to amplify target sequences for downstream cloning applications, including ligation, Cold Fusion, Gateway cloning, Gibson and Golden Gate assembly

Cultivation of E. coli and plasmid DNA isolation

Bacterial transformation with plasmid DNA, liquid culture cultivation, and manual or semi-automated plasmid DNA preparation at mini, midi, and maxi scale

DNA quality assessment

Assessment of DNA quantity and quality using NanoDrop and Qubit, restriction digestion followed by agarose gel electrophoresis, and evaluation of Sanger and Oxford Nanopore Technologies (ONT) sequencing results

Plasmid verification

In-house NGS library preparation and plasmid DNA analysis using an Illumina MiSeq sequencer and a custom-built analysis pipeline

Linearization service

Preparation of plasmid DNA for downstream RNA synthesis via in vitro transcription (IVT), including linearization and purification of DNA templates using the KingFisher Duo Prime Purification System

Publications

200 days ago, TRON took a decisive step toward advancing clinical translation by introducing the new role of Medical Director and appointing Univ.-Prof. Dr. Matthias Gaida. This move reflects our clear commitment to aligning research with clinical reality and patient care.

Why does this matter?
As Medical Director, Matthias ensures that TRON’s scientific strategy addresses real-world medical challenges. By integrating clinical perspectives into decision-making, research questions become more relevant, and promising discoveries can move faster into application.

What drives this approach?
A strong belief that science should serve patients. The goal is to accelerate translational research and strengthen collaborations—locally, nationally, and internationally. This includes building structured partnerships, developing early-stage study concepts, and ensuring access to clinical data and samples.

What’s next?
Under Matthias’ leadership, TRON is creating flexible pipelines for pilot projects and fostering research that responds directly to patient needs. This strategy will position TRON as a leading center for personalized immuno-oncology—while exploring new areas such as cardiology, where scientific discovery and patient care go hand in hand.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Gene Transfer Unit

  • Maximilian Mustermann

    Director Functional Unit

  • Maximilian Mustermann

    Deputy Director Functional Unit

Our activities and competences

Our methods and platforms

Trans-amplifying RNA

Together with the Vectors Unit we developed trans-amplifying RNA, our proprietary variant of self-amplifying RNA. Suitable to reduce the amount of antigen coding RNA needed for vaccination. Detailed description of scientific background and methods:

Production of retroviral particles and generation of cell lines

A technology that utilize modified retroviruses to deliver genes into cells for permanent expression. Gene transfer is mediated via viral particles. The vector system allows efficient but random insertion of genetic material with the size up to ~10 kb in vast majority of cells.

CRISPR/Cas9 knockout

CRISPR-Cas9 is a transformative gene-editing technology that revolutionizes genomic modifications because it allows precise genome editing directly within cells. The system consists of a short, non-coding guide RNA (gRNA) and the Cas9 nuclease, which together act as a genetic-engineering tool capable of disabling, repairing, or introducing new genetic material.

CRISPRa/i (activation/inhibition)

CRISPRa/i is based on Cas9 mutant which is not able to cut the DNA, so called dead Cas9 (dCas9). Fusing one or more transcription factors to dCas9 in combination with gRNAs targeting promotor regions turns the system into a tool suitable to specifically up- or downregulate expression of desired genes.