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1.
Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.  相似文献   

2.
Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.  相似文献   

3.
Interferon-α (IFN-α) is used clinically to treat hepatocellular carcinoma (HCC), although the detailed therapeutic mechanisms remain elusive. In particular, IFN-α has long been implicated in control of the cell cycle, but its actual point of action has not been clarified. Here, using time lapse imaging analyses of the human HCC cell line HuH7 carrying a fluorescence ubiquitination-based cell cycle indicator (Fucci), we found that IFN-α induced cell cycle arrest in the G0/G1 phases, leading to apoptosis through an IFN-α type-2 receptor (IFNAR2)-dependent signaling pathway. Detailed analyses by time lapse imaging and biochemical assays demonstrated that the IFN-α/IFNAR2 axis sensitizes cells to apoptosis in the S/G2/M phases in preparation for cell death in the G0/G1 phases. In summary, this study is the first to demonstrate the detailed mechanism of IFN-α as an anticancer drug, using Fucci-based time lapse imaging, which will be informative for treating HCC with IFN-α in clinical practice.  相似文献   

4.
Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.  相似文献   

5.
A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015. © 2015 Wiley Periodicals, Inc.  相似文献   

6.
7.
Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low‐density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin‐expressing cells that have been transduced by a Lodavin‐containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock‐in Lodavin mouse to develop biotin‐based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed βgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line‐derived R26RLodavin mouse line was generated. Efficient removal of the floxed βgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7‐Cre, Wnt4‐Cre, or Tie1‐Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction. genesis 50:693–699, 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

8.
We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010. © 2009 Wiley‐Liss, Inc.  相似文献   

9.
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, CreERT2, from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2nEGFP‐CreERT2/+ and TnEGFP‐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.  相似文献   

10.
Neural crest cells (NCCs) are a multipotent embryonic cell population that contributes to the formation of various craniofacial structures including teeth. It has been generally believed that dental enamel is an ectodermal derivative, whereas the dentin–pulp complex and the surrounding supporting tissues originate from NCC-derived mesenchyme. These traditional concepts stem mainly from several early studies of fishes and amphibians. Recently, Wnt1-Cre/R26R mice, a mouse model for NCC lineage analysis, revealed the contribution of NCCs to mammalian tooth development. However, the discrepancy of expression patterns between different NCC-specific transgenic mouse lines makes it compulsory to revisit the cell lineage in mammalian tooth development. Here, we reevaluated the NCC lineage during mouse tooth development by using P0-Cre/R26R mice, another NCC-specific transgenic mouse line. Inconsistent with the traditional concepts, we observed the potential contribution of NCCs to developing enamel organ and enamel formation. We also demonstrated that the P0-Cre transgene was specifically expressed in migrating NCC in the hindbrain region, where NCC contributes to tooth, validating their applicability for NCC lineage analysis. Our unanticipated finding may change the general understanding of tooth development and provide new insights into dental stem cell biology.  相似文献   

11.
12.
In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.  相似文献   

13.
14.
While cell proliferation is an essential part of embryonic development, cells within an embryo cannot proliferate freely. Instead, they must balance proliferation and other cellular events such as differentiation and morphogenesis throughout embryonic growth. Although the G1 phase has been a major focus of study in cell cycle control, it is becoming increasingly clear that G2 regulation also plays an essential role during embryonic development. Here we discuss the role of Cdc25, a key regulator of mitotic entry, with a focus on several recent examples that show how the precise control of Cdc25 activity and the G2/M transition are critical for different aspects of embryogenesis. We finish by discussing a promising technology that allows easy visualization of embryonic and adult cells potentially regulated at mitotic entry, permitting the rapid identification of other instances where the exit from G2 plays an essential role in development and tissue homeostasis.  相似文献   

15.
Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission‐fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far‐red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R‐mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.  相似文献   

16.
Several genetically modified mouse models have been generated in order to drive expression of the Cre recombinase in the neuroectoderm. However, none of them specifically targets the posterior neural plate during neurulation. To fill this gap, we have generated a new transgenic mouse line in which Cre expression is controlled by a neural specific enhancer (NSE) from the Caudal‐related homeobox 2 (Cdx2) locus. Analyses of Cre activity via breeding with R26R‐YFP reporter mice have indicated that the Cdx2NSE‐Cre mouse line allows for recombination of LoxP sites in most cells of the posterior neural plate as soon as from the head fold stage. Detailed examination of double‐transgenic embryos has revealed that this novel Cre‐driver line allows targeting the entire posterior neural tube with an anterior limit in the caudal hindbrain. Of note, the Cdx2NSE regulatory sequences direct Cre expression along the whole dorso‐ventral axis (including pre‐migratory neural crest cells) and, accordingly, YFP fluorescence has been also observed in multiple non‐cranial neural crest derivatives of double‐transgenic embryos. Therefore, we believe that the Cdx2NSE‐Cre mouse line represents an important novel genetic tool for the study of early events occurring in the caudal neuroectoderm during the formation of both the central and the peripheral nervous systems. genesis 51:777–784. © 2013 Wiley Periodicals, Inc.  相似文献   

17.
New sesquiterpene quinones, metachromins X (1) and Y (2), together with the known metachromins C (3), J (4), and T (5), were isolated as inhibitors of cell cycle progression in the HeLa/Fucci2 cells. The structure of 1 was assigned by spectroscopic data and confirmed by a total synthesis. The planar structure of 2 was determined by interpretation of spectroscopic data, whereas its absolute configuration was analyzed by a combination of chiral HPLC and CD spectroscopy. Metachromins X (1) and C (3) arrested the cell cycle progression of HeLa/Fucci2 cells at S/G2/M phase.  相似文献   

18.
Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

19.
The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ~ 84% of each lineage was EYFP+, and nearly all cells that come from Tie2‐expressing lineages are CD45+, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2+ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

20.
Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐RafAVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the BrafAVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, BrafAVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐RafAVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.  相似文献   

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