Transgenic and mutant mice

The transgenic (mouse mutant core) facility provides services mainly to the ISREC and local research community. These services include pronuclear injections of constructs to generate classical transgenic mice and blastocyst injections of embryonic stem cells, which are mainly used to generate knock-out mice. The introduction of genetic material into the germ line of mammals is one of the major experimental achievements developed in the last three decades, and genetically-engineered mice represent a powerful tool in today's biomedical research. To cope with the increasing demand for mouse studies and transgenic experiments, a transgenic facility has been set up at the ISREC/BIL already in 1992, providing pronuclear injection of DNA constructs and the injection of ES cells into blastocysts.

Pronuclear microinjection of DNA into fertilized mouse oocytes was the service most extensively used (Fig. 1). A DNA construct is provided by the scientist, and is injected into fertilized oocytes derived from either FVB/n or B6D2F1 hybrid mice. Integration of the DNA construct is random, and the number of copies might vary from one to several hundred, arranged most often in tandem head-to-tail arrays. At weaning of offspring, further analysis is performed by the scientist. We have also performed transient transgenic experiments, without need for establishing stable lines, and have reintroduced transgenes directly into genetically-deficient (knock-out) mice. If desired, the facility might collaborate and provide expertise in designing the construct and performing the analysis of the mice. We are injecting column-purified fragments isolated from plasmid vectors, but have also successfully generated mice following injection of supercoiled BAC DNA. Generation of chimeric mice by introducing embryonic stem (ES) cells into preimplantation embryos (mostly blastocysts) is an alternative approach to produce genetically modified mice. ES cell work is performed by the collaborating scientist, and the facility is then injecting correctly targeted ES cell clones into C57BL/6 host blastocysts. Resulting chimeras are then further bred by the scientist.

Figure 1: Generation of transgenic mice by pronuclear injection.

Pigment cells and melanoma

Research activities within the laboratory focus on regulation and biology of pigment cells. These comprise neural crest-derived melanocytes or cells of the retinal pigment epithelium (RPE). Pigment cells are important for skin, hair and eye pigmentation and thus implicated in diseases like albinism or melanoma. Our recent effort has focused on the generation and characterization of a new mouse model for melanoma, and on the impact of distal regulatory elements in regulating gene expression of pigmentation genes. Moreover, we are currently performing conditional knockout experiments to address gene function specifically in melanocytes.

Activation of N‑Ras is one of the most frequent oncogenic alterations found in human melanoma. To mimic this disease in mice, we have targeted expression of a dominant-active human N‑Ras (N‑Ras^Q61K) gene to melanocytes. Upon additional inactivation of the INK4a locus, encoding the tumor suppressors p16^INKA and p19^ARF, hyperpigmented N‑ras mice develop cutaneous melanoma (Fig. 2). Thus, melanoma highly similar to human disease can be modeled in mice by recapitulating the human mutations. This model can be used to elucidate mechanisms of melanoma progression and metastasis formation, and further may be used for preclinical testing of novel therapies. We have used this model to test implication of fibroblast growth factor 2 (Fgf2) which has been assigned a role in melanocyte proliferation and in development of human cutaneous melanoma. Our results suggested that, under conditions of active N‑ras, Fgf2 is not required for melanocyte proliferation and melanoma genesis.

Figure 2: Tyr::N‑Ras^Q61K transgenic mice which are INK4a-deficient develop melanotic melanoma.

The genes of the tyrosinase family, tyrosinase, Tyrp1 and Dct are expressed both in melanocytes and the RPE. The regulation of this expression pattern is not only controlled by promoter-proximal cis-acting elements, but also by distal regulatory elements (Fig. 3). Such a distal regulatory element has been described at ‑12 kb in the tyrosinase gene, and operates as a melanocyte-specific enhancer but also protects from spreading of condensed chromatin. We therefore assumed that genetic elements responsible for Tyrp1 expression in melanocytes are equally located outside of the promoter. We successfully rescued the Tyrp1^b (brown) phenotype in transgenic mice with a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences. Since these mice were undistinguishable from black mice, we believed that regulatory elements responsible for expression in melanocytes are present in the BAC. Comparison of vertebrate Tyrp1 loci with the BAC sequence revealed several stretches of conserved noncoding sequences. When tested in transgenic mice or in cell culture with a reporter gene, one of them appears to positively regulate Tyrp1 expression in melanocytes. This suggests that specific enhancers have crucial roles in the tight regulation of expression of tyrosinase family genes in either melanocytes or RPE.

Figure 3: Different cis-acting elements are implicated in melanocyte and RPE-specific gene expression of the tyrosinase family genes tyrosinase (Tyr), Tyrp1 and Dct. DRE, distal regulatory element; prom, promoter region.

Keywords

Transgenic, melanocyte, melanoma