Since the advent of CRISPR technology, many gene modifications can be directly done in zygotes, eliminating the need of generating mice from ES cells by chimeras. However, more complex alterations like the exchange of larger DNA sequences (e.g. mouse to human) still are best done in ES cells. ES cells are also a valuable tool to study the genetic influence on cellular differentiation.

In addition to our own projects, we offer collaborations for the generation of genetically modified mice and murine ES cells. This includes, but is not limited to:

• Advice in construct design and experimental set up.
• Generation of transgenic mice by pro-nucleus injection.
• Generation of chimeric mice by injection of ES cells into blastocyst stage embryos or by aggregation.
• Culture, transfection and selection of murine ES cells.
• Derivation of ES cells from mutant mice.
• Differentiation of murine ES cells
• Genome editing (KO,KI) by CRISPR/Cas9 directly in murine zygotes or ES cells.
• As a service we also offer cryopreservation, rederivation and sanitation of mouse lines.

DAGMAR - Danish Genetically Modified Animal Resource -  https://dagmar.au.dk/

An example of neuronal differentiation of murine ES in culture (click photo for enlargement).

Neuronal differentiation is visualized by immunostaining or ß-III-tubulin or neurofilament (left and center). Presence and absence of SorCS2 is shown by anti-SorCS2 staining (right). Homozygous SorCS2 KO cells (bottom row) differentiate very poorly in culture despite their ability to do so in situ (not shown), picture by Abuzar Kaleem.

We have earlier observed that translation of the bHLH transcription factor TWIST1 is differentially regulated in the developing embryo. We identified a number of micro-RNAs that inhibit translation in cultured cells. Using CRISPR based techniques, we try to identify regulatory elements that are responsible for the translational inhibition in the embryo.

Septins are a family of filament forming proteins. Septin filaments, which are referred to as the fourth part of the cytoskeleton, consist of repeats of heteromeric octamers. Several Septins, are associated with a variety of tumors. This is particularly true for Septin9, which surprisingly seem to have both proto-oncogenic and tumor suppressor function. This dualistic function might be connected to different SEPTIN9 isoforms that have been shown to differentially direct the association of Septin filaments with microtubules and actin filaments.

We try to elucidate the impact of SEPTIN9 isoforms on the cellular localization and cell stability.

A homozygous Sept9 KO fibroblast reconstituted with an EGFP tagged Sept9_ia variant. The septin filaments (green) are arranged in a stress fiber (actin) like patteren. Microtubuli are stained in red.