A<i>cre</i>-transgenic mouse strain for the ubiquitous deletion of<i>loxP</i>-flanked gene segments including deletion in germ cells

Schwenk, Frieder; Baron, Udo; Rajewsky, Klaus

doi:10.1093/nar/23.24.5080

Cited by 1,199 publications

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“…Mice bearing this allele were transferred to the Walter and Eliza Hall Institute (WEHI) animal facility, where they were crossed with C57BL/6 deleter CMV-Cre mice 51 to breed out the FLPe transgene and to cause recombination at the loxP sites flanking A1-b to produce the constitutive knockout allele (A1 +/ − mice). A1 +/ − ; Cre T/+ mice were crossed with A1 +/ − mice in order to generate A1 −/− mice.…”

Section: Methodsmentioning

confidence: 99%

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

et al. 2017

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The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated. Cell Death and Differentiation (2017) 24, 534-545; doi:10.1038/cdd.2016.156; published online 13 Janaury 2017The pro-survival proteins of the BCL-2 family prevent apoptosis 1 and studies using gene-targeted mice have revealed which cell types rely on which pro-survival protein for their survival. For example, Bcl-2 −/− mice exhibit thymic and splenic atrophy, a loss of fur pigment and die~30 days post birth from polycystic kidney disease, attributable to excess lymphocyte, melanocyte and renal epithelial cell apoptosis, respectively. 2-4 Bcl-X −/− mice die before E14.5 of embryonic development because of aberrant death of erythroid and neuronal cells. 5 The generation of chimaeric or tissue-specific Bcl-X −/− revealed a critical role for BCL-XL in the survival of developing lymphocytes 5 and platelets. 6,7 Mcl-1 −/− embryos die before implantation (E3.5), 8 but conditional Mcl-1 deletion models have demonstrated an essential role for MCL-1 in the survival of haematopoietic stem cells, lymphocytes, neurons and cardiomyocytes. 9-15 Bcl-W −/− mice have impaired spermatogenesis. 16,17 A1 remains the only pro-survival BCL-2 family member for which a knockout mouse strain has not been developed. A1 was first discovered as a GM-CSF-inducible gene with significant sequence similarity to BCL-2 and MCL-1, 18 and its human homologue BFL-1 was later identified in fetal liver. 19 Overexpression of A1 protected an IL-3-dependent cell line from growth factor deprivation-induced apoptosis, thus demonstrating its pro-survival function. 20 In mice, A1 expression is restricted to the haematopoietic compartment. 18 Human BFL-1 expression is more widespread, but also predominantly haematopoietic. 21 A1 can be upregulated by NF-κB signalling, and it has been proposed that A1/BFL-1 is important for the survival of several activated immune cell subsets through stimulation of antigen or cytokine receptors. [22][23][24][25] Studies of A1 in mice are complicated by the presence of multiple isoforms that are the result of gene duplication ev...

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Section: Methodsmentioning

confidence: 99%

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

et al. 2017

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“…MR/X mice were generated on a C57Bl/6 background by breeding homozygous MR lox/lox males with homozygous MR lox/lox females in addition heterozygous for the Xchromosome linked cmv-cre [24]. All experiments were done in female mice homozygous for MR lox/lox and heterozygous for cmv-cre expression (MR/X).…”

Section: Animalsmentioning

confidence: 99%

“…Thus, mating of mice with a X-chromosome-linked expression of the cre recombinase [24] with mice with floxed MR alleles [21] generated female mice carrying a targeted deletion of MR in some, but not all, of their cells (MR/X mice). In this model, MR-positive (MR wt ) and MR-negative (MR ko ) cells can be studied next to each other in the same physiological context.…”

Section: Introductionmentioning

confidence: 99%

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Czogalla

Vohra

Pentón

et al. 2016

Pflugers Arch - Eur J Physiol

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Aldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na+ reabsorption via up-regulation of the epithelial Na+ channel (ENaC) and the Na+-K+-ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in 20% of renal tubule cells (MR/X mice), in which MR-positive (MRwt) and -negative (MRko) cells can be studied sideby-side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MRwt and MRko cells and dietary Na+ restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MRko cells in the CS did not show any detectable alpha-ENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na+ restriction. Furthermore, Na+-K+-ATPase expression was unaffected in MRko cells of the DCT, while it was lost in MRko cells of the CS. In conclusion, MR is crucial for ENaC and Na+-K+-ATPase regulation in the CS, but is dispensable for NCC and Na+-K+-ATPase regulation in the DCT. PAEJ-D-16-00005 -NCC regulation is MR independent 2 ACKNOWLEDGEMENTSThe authors would like to thank Günter Schütz and Stefan Berger, Burkhard Becher, Celso E. Gomez-Sanchez, and Eric Feraille for kindly providing us with the MRlox/lox mouse line, the cmv-cre mouse line, the anti-MR antibodies, and the anti-Na + -K + -ATPase antibody, respectively. The expert technical assistance by Monique Carrel and Michèle Heidemeyer is gratefully acknowledged. GRANTS DISCLOSURESThe authors declare no conflict of interest, financial or otherwise. PAEJ-D-16-00005 -NCC regulation is MR independent 3 ABSTRACTAldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na + reabsorption via up-regulation of the epithelial Na + channel (ENaC) and the Na + -K + -ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in ~20% of renal tubule cells (MR/X mice), in which MR-positive (MR wt ) and -negative (MR ko ) cells can be studied sideby-side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MR wt and MR ko cells and dietary Na + restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MR ko cells in the CS did not show any detectable alphaENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na + restriction. Furtherm...

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“…We generated Ano2 -/-mice by flanking exon 12 with loxP sites and crossing these mice with Cre-recombinase-expressing deleter mice 24 (Supplementary Fig. 1a,b).…”

Section: Disruption Of Ano2 and Ano2 Expression Patternmentioning

confidence: 99%

Ca2+-activated Cl− currents are dispensable for olfaction

et al. 2011

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Graduate student at the Freie Universität Berlin Olfaction in mammals involves the binding of odorants to a subset of the large number of different G protein-coupled receptors that are present in the cilia of olfactory sensory neurons (OSNs) 1 . These neurons are predominantly found in the MOE, which senses and discriminates myriad volatile compounds. In the mouse, the nose contains additional, apparently more specialized olfactory organs, such the septal organ of Masera and the VNO 2 . Sensory neurons in the VNO are morphologically distinct and use odorant receptors and signal transduction cascades that differ from those found in the MOE.In the canonical OSN signal transduction pathway, odorant binding to the receptor locally increases cytosolic cAMP through activation of the olfactory G protein G olf and adenylate cyclase type III 1,2 . cAMP then opens heteromeric cyclic nucleotide-gated (CNG) cation channels 3 . The resulting influx of Na + and Ca 2+ depolarizes the plasma membrane and raises the cytosolic Ca 2+ concentration ([Ca 2+ ] i ), thereby activating Ca 2+ -activated Cl -channels 1,4-10 . All these components of the signal transduction cascade are localized to sensory cilia, which are embedded in the mucus covering the MOE. These cilia provide a large interaction surface for odorants, and the cilia's small diameters facilitate large local increases in cytosolic cAMP and [Ca 2+ ].There has been broad consensus that Ca 2+ -activated Cl -channels powerfully amplify olfactory signal transduction 1,4-10 . These channels are thought to mediate an outward flow of Cl -, which generates a depolarizing current that, in rodents, is five-to tenfold larger than currents through CNG channels 8,11,12 . A prerequisite for Cl -efflux 3 is an inside-out electrochemical Cl --gradient. It is believed that cytosolic chloride concentration of OSNs is raised by the Na + K + 2Cl -co-transporter Nkcc1 9,10,13 and that [Cl -] is low in the mucus surrounding the cilia 14,15 . However, mucosal ion concentrations are difficult to measure in vivo, and Nkcc1 knockout mice display attenuated electro-olfactograms (EOGs) 11,16 , but normal olfactory sensitivity 17 .To investigate the role of Ca 2+ -activated Cl -channels in olfaction, we disrupted Ano2 in mice. Ano2 is a member of the Anoctamin (Tmem16) gene family, which encodes several Ca 2+ -activated Cl -channels [18][19][20] . Agreeing with recent results 13,21-23 , our knockout-controlled immunolabeling showed Ano2 expression in cilia of OSNs in the MOE, in microvilli of VNO sensory neurons and in synapses of photoreceptors. Additionally, we found Ano2 in the olfactory bulb. Ano1 expression overlapped with Ano2 in the VNO and the retina, but not in the MOE. Patch-clamp analysis showed that Ca 2+ -activated Cl -currents were undetectable in Ano2 -/-OSNs.Unexpectedly, however, EOGs of Ano2 -/-mice were reduced by only up to ~40%, and Ano2 -/-mice were able to smell normally. Our work calls for a revision of the current view that Ca 2+ -activated Cl -channels have a crucial role...

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Acre-transgenic mouse strain for the ubiquitous deletion ofloxP-flanked gene segments including deletion in germ cells

Cited by 1,199 publications

References 11 publications

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Ca2+-activated Cl− currents are dispensable for olfaction

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