Rapid generation of inducible mouse mutants

Seibler, Jost; Zevnik, Branko; Birgit, Küter-Luks; Andreas, Susanne; Kern, Heidrun; Hennek, Thomas; Rode, Anja; Heimann, C.; Faust, Nicole; Kauselmann, Gunther; Schoor, Michael; Jaenisch, Rudolf; Rajewsky, Klaus; Kühn, Ralf; Schwenk, Frieder

doi:10.1093/nar/gng012

Cited by 279 publications

(281 citation statements)

References 26 publications

(22 reference statements)

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“…Because CAG::creER T2 mice have not been reported and because of the above factors that make meaningful comparisons between different transgenic lines difficult, it remains to be seen how FlpeER T2 precisely compares to CreER T2 in these types of activity assays in embryos. While CAG::creER T2 mice have not been described, ROSA26::creER T2 mice have; however, the assays reported are restricted to adult as opposed to embryonic tissues: in many adult organs, excision of a ROSA26 target allele was observed in *30-60% of cells following five consecutive daily doses of 5 mg of TAM (Seibler et al, 2003). It should be noted that whether using CreER or FlpeER fusions, the extent of recombination following induction depends greatly on the level of fusion protein produced and therefore on the robustness of the employed driver elements coupled with transgene integration site and copy number.…”

Section: Discussionmentioning

confidence: 99%

Ligand-activated Flpe for temporally regulated gene modifications

Hunter

Awatramani

Farley

et al. 2005

genesis

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Summary:The selectivity by which site-specific recombinase-mediated genetic changes can be targeted to specific cells in the mouse has been limited by the fact that many genes used as recombinase ''drivers'' are expressed either in cell populations that change over time or constitutively in a given cell population for an extended time period, for example, in a germinal zone that gives rise successively to different lineages. These scenarios limit the selective dimension of conditional gene modification experiments as they preclude studying the later-generated lineages either because of earlier phenotypes (in the case of conditional mutagenesis experiments) or because the early and permanent activation of a reporter in a germinal zone results in all descendant lineages being marked (in the case of fate-mapping experiments). To circumvent this limitation, inducible forms of Cre recombinase have been developed, enabling the induction of genetic changes in late embryonic or adult cells accessible only through late aspects of a dynamic driver gene expression profile. To increase the number of tools available for engineering genetic changes in selective cell populations, we have generated a ligand-regulated form of Flpe using the recombinase-steroid receptor fusion approach. In two prototypical scenarios, we show that the fused gene product, FlpeER T2 , is competent to mediate DNA recombination in vivo and responds specifically to the inducer tamoxifen in a dose-dependent manner without detectable background activity. genesis 41:99-109, 2005.

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Section: Discussionmentioning

confidence: 99%

Ligand-activated Flpe for temporally regulated gene modifications

Hunter

Awatramani

Farley

et al. 2005

genesis

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“…One line of R26CreER T2 mice was purchased from ARTEMIS Pharmaceuticals (17). Another line of R26CreER T2 mice was generated by Regeneron Pharmaceuticals using Velocigene technology (25), essentially as described.…”

Section: Animal Usementioning

confidence: 99%

“…The fusion protein is inactivated by binding to heat shock proteins, until the administration of TM, when it is released from the complex, becomes active and excises loxPflanked DNA regions. Several transgenic mouse lines have been generated that express CreER T2 fusion genes under the control of tissue-specific promoters, which show ligand-dependent recombination in certain cell types (12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2

Higashi¹,

Ikawa

Muramatsu

et al. 2009

The Journal of Immunology

128

119

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The CreER T2 for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER T2 ) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER T2 in adult mice and embryos. The toxicity of Cre recombinase or CreER T2 in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER T2 is knocked-in into the Rosa26 locus (R26CreER T2 mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER T2 mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER T2 toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER T2 on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER T2 in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER

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“…For the staining of PIP 3 (phosphatidylinositol-3,4,5 triphosphate) in POMC neurons, Stat3-C POMC mice were crossed with ROSAArte26 reporter mice (Seibler et al, 2003). At the age of 12 weeks, 16-h-fasted animals were anesthetized and intravenously injected with 5 U of human regular insulin (Novo Nordisk) for 10 min.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…For X-gal stainings, Stat3-C POMC were mated with ROSAArte26 reporter mice (Seibler et al, 2003). At the age of 12 weeks, 16-h-fasted animals were anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4).…”

Section: Combined Histochemistry and In Situ Hybridizationmentioning

confidence: 99%