A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

A series of Cre‐ERT2 drivers for manipulation of the skeletal muscle lineage

We report the generation of five mouse strains with the tamoxifen‐inducible Cre (Cre‐ER^T2; CE) gene cassette knocked into the endogenous loci of Pax3, Myod1, Myog, Myf6, and Myl1, collectively as a resource for the skeletal muscle research community. We characterized these CE strains using the Cre reporter mice, R26R^LacZ, during embryogenesis and show that they direct tightly controlled tamoxifen‐inducible reporter expression within the expected cell lineage determined by each myogenic gene. We also examined a few selected adult skeletal muscle groups for tamoxifen‐inducible reporter expression. None of these new CE alleles direct reporter expression in the cardiac muscle. All these alleles follow the same knock‐in strategy by replacing the first exon of each gene with the CE cassette, rendering them null alleles of the endogenous gene. Advantages and disadvantages of this design are discussed. Although we describe potential immediate use of these strains, their utility likely extends beyond foreseeable questions in skeletal muscle biology. genesis 52:759–770, 2014. © 2014 Wiley Periodicals, Inc.     

Medial calcification in the arterial wall of smooth muscle cell‐specific Smpd1 transgenic mice: A ceramide‐mediated vasculopathy

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1^trg/SM^cre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1^trg/SM^wt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1^trg/SM^cre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1^trg/SM^cre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1^trg/SM^cre mice, increased P_i concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in P_i‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.     

Use of FOXJ1CreER2T mice for inducible deletion of embryonic node gene expression

The ciliated cells of the node of the mouse embryo contribute to the establishment of left–right patterning via generation of leftward laminar fluid flow and initiation of a left‐sided morphogen gradient. Here, we identify FOXJ1CreER^2T mice in which expression of Cre recombinase is directed to ciliated node cells. The data demonstrate that foxj1 is expressed specifically in the node throughout the developmental window critical for left–right patterning. In transgenic embryos, Cre expression is detected by immunohistochemistry in ciliated cells of the node. Rosa26R reporter mice, in which expression of lacZ is activated only after Cre‐mediated recombination, demonstrate strong and uniform labeling at the node when crossed with FOXJ1CreER^2T mice. Cell labeling occurred as early as 0‐ to 2‐somite stages, specifically within cells of the node, and recombination was highly efficient in response to tamoxifen. FOXJ1CreER^2T transgenic mice represent a new genetic tool for the analysis of node‐specific gene expression and will also be valuable in the study of node cell lineage and temporal cell fate mapping. genesis 47:132–136, 2009. © 2009 Wiley‐Liss, Inc.     

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC‐specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand‐binding domain (ER^T2). In the background of the Cre reporter mouse strain Rosa26^lacZ, we show that the fusion CreER^T2 recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by β‐galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs. genesis 47:122–131, 2009. © 2008 Wiley‐Liss, Inc.     

Tamoxifen‐inducible Cre‐mediated recombination in adipocytes

To generate a mouse line which allows inducible, Cre/loxP‐dependent recombination in adipocytes, we used RedE/RedT‐mediated recombineering to insert the CreER^T2‐transgene, which encodes a fusion protein of Cre and a mutated tamoxifen‐responsive estrogen receptor, into the start codon of the adipocyte‐specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER^T2 mouse line (97%–99%), while no recombination was seen in vehicle‐treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER^T2 in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER^T2 mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue. genesis 48:618–625, 2010. © 2010 Wiley‐Liss, Inc.     

A Cre knock‐in mouse line on the Sickle tail locus induces recombination in the notochord and intervertebral disks

Sickle tail (Skt) was originally identified by gene trap mutagenesis in mice, and the trapped gene is highly expressed in the notochord, intervertebral discs (IVD), and mesonephros. Here, we report the generation of Skt^cre mice expressing Cre recombinase in the IVD due to target insertion of the cre gene into the Skt locus by recombinase‐mediated cassette exchange. Crossing a conditional lacZ Reporter (R26R), Cre expression from the Skt^cre allele specifically activates β‐galactosidase expression in the whole notochord from E9.5 onwards. In E15.5 Skt^cre;R26R embryos, reporter activity was detected in the nucleus pulposus and in a portion of the annulus fibrosus, resulting in expansion of Cre‐expressing cells in the adult IVD. Reporter activity was also seen in the Skt^cre;R26R mesonephros at E15.5. These results suggest that Skt^cre mice are useful for exploring the fate specification of notochordal cells and creating models for IVD‐related skeletal diseases. genesis 50:758–765, 2012. © 2012 Wiley Periodicals, Inc.     

A mouse tool for conditional mutagenesis in ovarian granulosa cells

Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER^T2 fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17‐beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1‐iCreER^T2 transgenic mice express the iCreER^T2 fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with “floxed” Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1‐iCreER^T2 mice using a Cre reporter line demonstrated that Cre‐mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1‐iCreER^T2 mice should be a useful tool to analyze the gene functions in ovarian granulosa cells. genesis 48:612–617, 2010. © 2010 Wiley‐Liss, Inc.     

Highly B lymphocyte‐specific tamoxifen inducible transgene expression of CreERT2 by using the LC‐1 locus BAC vector

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio‐temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte‐specific tamoxifen‐inducible Cre transgenic mouse strain, LC‐1‐hCD19‐CreER^T2. We utilized the human CD19 promoter for expression of the tamoxifen‐inducible Cre recombinase (CreER^T2) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross‐breeding the LC‐1‐hCD19‐CreER^T2 strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC‐1‐hCD19‐CreER^T2 strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse. genesis 47:729–735, 2009. © 2009 Wiley‐Liss, Inc.     

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/<Emphasis Type="Italic">lox</Emphasis>P strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T₀ plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T₀ transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T₀-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T₂ seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T₁ plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Generation of knock‐in mice that express nuclear enhanced green fluorescent protein and tamoxifen‐inducible Cre recombinase in the notochord from Foxa2 and T loci

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time‐lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock‐in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen‐inducible Cre, CreER^T2, from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2^{nEGFP‐CreERT2/+} and T^{nEGFP‐CreERT2/+} embryos, nuclei of the Foxa2 or T‐expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. genesis 51:210–218, 2013. © 2013 Wiley Periodicals, Inc.     

Enhanced Rate Capability of Ion‐Accessible Ti3C2Tx‐NbN Hybrid Electrodes

Although 2D Ti₃C₂T_x is a good candidate for supercapacitors, the restacking of nanosheets hinders the ion transport significantly at high scan rates, especially under practical mass loading (>10 mg cm^?2) and thickness (tens of microns). Here, Ti₃C₂T_x‐NbN hybrid film is designed by self‐assembling Ti₃C₂T_x with 2D arrays of NbN nanocrystals. Working as an interlayer spacer of Ti₃C₂T_x, NbN facilitates the ion penetration through its 2D porous structure; even at extremely high scan rates. The hybrid film shows a thickness‐independent rate performance (almost the same rate capabilities from 2 to 20 000 mV s^?1) for 3 and 50 µm thick electrodes. Even a 109 µm thick Ti₃C₂T_x‐NbN electrode shows a better rate performance than 25 µm thick pure Ti₃C₂T_x electrodes. This method may pave a way to controlling ion transport in electrodes composed of 2D conductive materials, which have potential applications in high‐rate energy storage and beyond.     

Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3–C4 intermediate photosynthetic phenotype

In order to establish a model system for comparative studies of C₃ and C₃–C₄ intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C₃–C₄ photosynthetic intermediate species M. arvensis and the C₃ species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the β‐glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium‐mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter‐driven, modified version of gfp, the mgfp5‐ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819–832). GFP5 produced bright‐green fluorescence in transformed tissues that was distinctly detected 5–12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5‐ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T₁ and T₂ progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T₀) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5‐ER or gusA reporter genes were expressed in 90–95% of the kanamycin‐resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines.     

Streptomyces phytohabitans sp. nov., a novel endophytic actinomycete isolated from medicinal plant Curcuma phaeocaulis

A novel actinomycete, designated strain KLBMP 4601^T, was isolated from the root of the medicinal plant Curcuma phaeocaulis collected from Sichuan Province, south-west China. The strain produced extensively branched substrate and aerial hyphae that carried straight to flexuous spore chains. Chemotaxonomic properties of this strain were consistent with those of members of the genus Streptomyces. The cell wall of strain KLBMP 4601^T contained ll-diaminopimelic acid as the characteristic diamino acid. The major menaquinone was MK-9(H₄), with minor amounts of MK-9(H₆), MK-9(H₈) and MK-10(H₂). The major fatty acids were C_16:0, iso-C_16:0, C_18:1ω9c and C_16:1, iso G. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain KLBMP 4601^T belongs to the genus Streptomyces and is most closely related to Streptomyces armeniacus JCM 3070^T (97.9 %), Streptomyces pharmamarensis PM267^T (97.6 %) and Streptomyces artemisiae YIM 63135^T (97.5 %). The 16S rRNA gene sequence similarity between strain KLBMP 4601^T and other members of this genus were lower than 97.5 %. DNA–DNA hybridization studies of strain KLBMP 4601^T with the three closest species showed relatedness values of 36.3 ± 4.2 %, 27.3 ± 0.6 %, and 30.9 ± 2.5 %, respectively. On the basis of chemotaxonomic, phenotypic and genotypic characteristics, it is evident that strain KLBMP 4601^T represents a novel species of the genus Streptomyces, for which the name Streptomyces phytohabitans sp. nov. is proposed. The type strain is KLBMP 4601^T (=KCTC 19892^T = NBRC 108772^T).     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.