Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

Abstract

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination-excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ER T ), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ER T under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (Ϸ40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ER T in a given cell type, we have now crossed Cre-ER T -expressing mice with reporter mice in which expression of Escherichia coli ␤-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6-to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ER T . These results indicate that cell-specific expression of Cre-ER T in transgenic mice can be used for efficient tamoxifendependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

“…The R26R reporter has thus far proven to be an ideal reporter because of its expression in embryonic and adult cells, including those of the central and peripheral nervous systems, limb, skin, lung, and heart. A significant advance in genetic fate mapping has been the introduction of inducible forms of Cre in mice to accurately control when cells are marked (Brocard et al, 1997;Kimmel et al, 2000;Zirlinger et al, 2002;Guo et al, 2003;Ahn and Joyner, 2004;Harfe et al, 2004;Zervas et al, 2004). We have termed such an approach Genetic Inducible Fate Mapping, or GIFM (Fig.…”

“…1). The most common strategy for temporally regulating Cre activity has been to generate Cre fusion proteins with a tamoxifen-responsive Estrogen Receptor ligand binding domain from the human (ER T , Feil et al, 1996 andBrocard et al, 1997;ER T2 , Feil et al, 1997) or mouse (ER TM , Hayashi and McMahon, 2002) gene. CreER is sequestered in the cytoplasm by heat shock protein 90 (Hsp 90).…”

Kinetics of tamoxifen‐regulated Cre activity in mice using a cartilage‐specific CreERT to assay temporal activity windows along the proximodistal limb skeleton

“…Tamoxifen-regulated CreER T transgenic lines (Feil et al, 1996;Brocard et al, 1997;Vooijs et al, 2001;Hayashi and McMahon, 2002; reviewed by Nagy, 2000) offer versatile tools to dissect the different functions of genes that play critical roles at multiple stages of development and postnatally and, thus, have the potential to greatly increase our understanding of the regulation of tissues and cell types that form over a broad developmental time range. They have also proven to be very powerful tools for lineage and fate mapping studies in mice (e.g., Kimmel et al, 2000;Harfe et al, 2004;Ahn and Joyner, 2004).…”

Of mice and models: improved animal models for biomedical research

“…The first brand of inducible Cre recombinases employs a truncated progesterone receptor which is directly fused to the Cre recombinase (96,157), whereas the second approach makes use of a fusion between the Cre recombinase and an estrogen receptor (48, 93, 94, 116, 124, 127). Nevertheless, close inspection of the induced recombination frequencies clearly demonstrated that recombination mediated by some first-generation fusion-recombinases might be mosaic depending very much on the tissue type (13,24,67,124,143,145). It is therefore to be assumed that better inducible recombinase molecules are needed to efficiently regulate Cre or Flp activity in the mouse (57,59,85,157).…”