Gene Manipulation

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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.

Dr. Melanie Gräßl

Co-Director Gene Manipulation
Dr. Mario Perkovic

Co-Director Gene Manipulation

Our vision

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

Cloning

Dr. Melanie Gräßl

Head of Functional Unit Cloning
Ivica Fucek

Deputy Head of Functional Unit Cloning

Our activities and competences

Molecular cloning of plasmid DNA

State-of-the-art cloning services, continuously improved and offered either individually or as a complete package
Plasmid verification and quality control

Quality assessment, sequence analysis, and evaluation using next-generation sequencing (NGS)
Design and sequence optimization

Support with plasmid design, cloning strategies, and nucleotide optimization methods
Preparation of DNA templates for IVT

Linearization and purification of DNA templates to enable downstream RNA synthesis via in vitro transcription (IVT)

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

Gene Transfer

Dr. Mario Perkovic

Head of Functional Unit Gene Transfer

Our activities and competences

Gene transfer

Packaging and transmission to target cells with viral particles
ARISE

EU-funded project to establish industrial scale bioproduction of mRNA in yeast
Gene manipulation

Editing of genes via CRISPR/ Cas system for knockout or activation/inhibition (CRISPRa/i)
Chikungunya vaccine

Exploitation of taRNA technology to generate Chikungunya vaccine
Innate Immunity

Characterization and fine-tuning of innate immune responses to RNA vectors

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.