Following the Cycle: Finally, a Transgenic Mouse to Sort Live Replicating Cells

Department of Genetics and Institute for Diabetes, Obesity & Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: .
Developmental Cell (Impact Factor: 9.71). 10/2012; 23(4):676-7. DOI: 10.1016/j.devcel.2012.10.006
Source: PubMed


Studies of the mammalian cell cycle in vivo have been hampered by the lack of pure populations of proliferating cells. In this issue of Developmental Cell, Klochendler and colleagues (2012) develop a novel transgenic mouse that expresses Cyclin B1-GFP ubiquitously. By sorting and analyzing proliferating hepatocytes, they provide evidence for their transient dedifferentiation.

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    ABSTRACT: Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.
    Developmental Cell 10/2012; 23(4):681-690. DOI:10.1016/j.devcel.2012.08.009 · 9.71 Impact Factor
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    ABSTRACT: The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.
    Molecular Biology of the Cell 03/1995; 6(2):185-97. DOI:10.1091/mbc.6.2.185 · 4.47 Impact Factor
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    ABSTRACT: The molecular analysis of mammalian cellular proliferation in vivo is limited in most organ systems by the low turnover and/or the asynchronous nature of cell cycle progression. A notable exception is the partial hepatectomy model, in which quiescent hepatocytes reenter the cell cycle and progress in a synchronous fashion. Here we have exploited this model to identify regulatory networks operative in the mammalian cell cycle. We performed microarray-based expression profiling on livers 0-40 h post-hepatectomy corresponding to G0, G1, and S phases. Differentially expressed genes were identified using the statistical analysis program PaGE (Patterns from Gene Expression), which was highly accurate as confirmed by quantitative reverse transcription-PCR of randomly selected targets. A shift in the transcriptional program from genes involved in lipid and hormone biosynthesis in the quiescent liver to those contributing to cytoskeleton assembly and DNA synthesis in the proliferating liver was demonstrated by biological theme analysis. In a novel approach, we employed computational pathway analysis tools to identify specific regulatory networks operative at various stages of the cell cycle. This allowed us to identify a large cluster of genes controlling mitotic spindle assembly and checkpoint control at the 40-h time point as regulated at the mRNA level in vivo.
    Journal of Biological Chemistry 03/2005; 280(5):3715-22. DOI:10.1074/jbc.M410844200 · 4.57 Impact Factor

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