Testing and improving transgenic technologies

Transgenic techniques have allowed the modification of the mouse genome since its first implementation in 1982. The tools that can now be used to understand the physiology of the mouse range from simple overexpression transgenes, still an important technique today, to the use of recombinases such as the Cre/loxP, Dre/rox and FLP/frt systems to enable lineage tracing and cell-type specific knockouts. In the past, we have not only utilized these for our research but also generated tools useful for the broader community such as the Cre-inducible diphtheria toxin receptor in the iDTR strain, in which the Cre-technology was used to allow DTR expression in specific cell types, which can then be ablated by injection of diphtheria toxin (Buch et al. 2005), or the CD4-CreERt2 strain which can be used for tamoxifen-induced gene inactivation in T helper cells (Sledzinska et al. 2013). Transgenic techniques have been continuously developed further. We have been among the first to use a first generation designer nuclease technology, zinc finger nucleases, for direct targeted gene modification in the mouse oocyte (Hermann et al. 2012). After further experience with Tal effector nucleases (TALEN) we have now embraced fully the Cas9/CRISPR technology. Its ease of use makes it a valuable tool for in vivo analyses in the mouse. Beyond generation of simple deficiency alleles through generation of insertions and deletions, we have introduced point mutations by use of recombination substrates - all directly in the mouse oocyte. We are currently evaluating the use of Cas9/CRISPR for direct cell type specific mutagenesis, a quick-and-dirty replacement of the Cre/loxP system. Also, we are testing the Cas9/CRISPR system for use in transcriptional control, enabling the defined transcriptional activation and repression of scientifically interesting target genes. A further technological research topic is the improvement of in vivo monitoring of tumor growth in combination with live assessment of tumor infiltrating lymphocytes.