Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional sitespecific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.The study of the genetic control of mammalian development and physiology has been revolutionized by the ability to inactivate (knockout) specific genes by homologous recombination in the mouse (1). However, using current gene targeting technology, interpretations of knockout phenotypes are often limited by several factors. First, the presence of a selection marker may influence the phenotype of the mutation (2, 3). Second, artefacts can arise due to the lack of a gene product for the whole lifetime of the animal. Third, the inactivation of a gene may result in intra-utero lethality, precluding analysis of the possible function(s) of the gene at later stages of development and/or post-natally. A conditional gene targeting method based on the inducible activity of an engineered DNA recombinase could overcome these limitations by allowing the removal of the selection cassette and the timed and tissuespecific inactivation of target genes at will during development and in the adult mouse (4). Furthermore, such an inducible system could help in certain cases to distinguish between anomalies related to a mixed genetic background and those due to mutation of the targeted gene.The bacteriophage P1 Cre recombinase efficiently excises DNA flanked by two directly repeated loxP recognition sites in mammalian cells (5, 6). We have previously reported that fusion of the ligand-binding domain (LBD) of the estrogen receptor (ER) to the Cre recombinase generates a chimeric recombinase whose activity in cultured cells is dependent on the presence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) (7). To achieve conditional gene targeting in mice, where endogenous estradiol is present, we have subsequently fused Cre to a mutated LBD of the human ER (Gly 521 -> Arg, G521R) resulting in the chimeric protein Cre-ERT. Indeed, the corresponding mouse ER LBD mutant (G525R) does not bind 17,B-estradiol (E2), whereas it binds the synthetic ligands tamoxifen and 4-hydroxytamoxifen (OHT) (8). We report here that Cre-ERT is a functional tamoxifen-dependent recombinase in cultured cells and in transgenic m...

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

Mitochondria are essential to maintain neuronal viability. In addition to the generation of ATP and maintenance of calcium homeostasis, the effective delivery of mitochondria to the appropriate location within neurons is also likely to influence their function. In this study we examined mitochondrial movement and morphology in primary cultures of rat forebrain using a mitochondrially targeted enhanced yellow fluorescent protein (mt-eYFP). Mt-eYFP-labeled mitochondria display a characteristic elongated phenotype and also move extensively. Application of glutamate to cultures results in a rapid diminution of movement and also an alteration from elongated to rounded morphology. This effect required the entry of calcium and was mediated by activation of the NMDA subtype of glutamate receptor. Treatment of cultures with an uncoupler or blocking ATP synthesis with oligomycin also stopped movement but did not alter morphology. Interestingly, application of glutamate together with the uncoupler did not prevent the changes in movement or shape but facilitated recovery after washout of the stimuli. This suggests that the critical target for calcium in this paradigm is cytosolic. These studies demonstrate that in addition to altering the bioenergetic properties of mitochondria, neurotoxins can also alter mitochondrial movement and morphology. We speculate that neurotoxin-mediated impairment of mitochondrial delivery may contribute to the injurious effects of neurotoxins.

In cells, stable microtubules are covalently modified by a carboxy-peptidase which removes the C-terminal tyrosine residue of α-tubulin. The significance of this selective detyrosination of microtubules is not understood. Here, we report that tubulin detyrosination in fibroblasts inhibits microtubule disassembly. This inhibition is relieved by overexpression of the depolymerizing motor MCAK. Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated microtubules in fibroblasts. Detyrosination of microtubules suppresses the activity of MCAK in vitro, apparently due to a decreased affinity of the ADP-Pi and ADP bound forms of MCAK for the microtubule lattice. Detyrosination also impairs microtubule disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that microtubule depolymerizing motors are directly inhibited by detyrosination of tubulin, resulting in stabilization of cellular microtubules. Detyrosination of transiently stabilized microtubules may give rise to persistent subpopulations of disassembly-resistant polymers to sustain sub-cellular cytoskeletal differentiation.