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Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice.

BMC biotechnology | Dec 24, 2004

BACKGROUND: Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. RESULTS: We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. CONCLUSIONS: Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate.

Pubmed ID: 15619330 RIS Download

Mesh terms: Animals | Cell Nucleus | Cell Proliferation | Embryo, Mammalian | Genes, Reporter | Green Fluorescent Proteins | Histones | Humans | Mice | Mice, Transgenic | Microscopy, Confocal | Mitosis | Nucleosomes | Recombinant Fusion Proteins | Stem Cells | Survival Rate

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Associated grants

  • Agency: NICHD NIH HHS, Id: R37 HD033082
  • Agency: NIGMS NIH HHS, Id: R01 GM060561
  • Agency: NIGMS NIH HHS, Id: GM60561
  • Agency: NICHD NIH HHS, Id: R01 HD033082
  • Agency: NICHD NIH HHS, Id: HD33082

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ImageJ

A Java image processing program which can display, edit, analyze, process, save and print 8-bit, 16-bit and 32-bit images. It can read many image formats including TIFF, GIF, JPEG, BMP, DICOM, FITS and raw. It runs, either as an online applet or as a downloadable application, on any computer with a Java 1.4 or later virtual machine. Downloadable distributions are available for Windows, Mac OS, Mac OS X and Linux. It supports stacks, a series of images that share a single window. It is multithreaded, so time-consuming operations such as image file reading can be performed in parallel with other operations. It can calculate area and pixel value statistics of user-defined selections. It can measure distances and angles. It can create density histograms and line profile plots. It supports standard image processing functions such as contrast manipulation, sharpening, smoothing, edge detection and median filtering. It does geometric transformations such as scaling, rotation and flips. Image can be zoomed up to 32:1 and down to 1:32. All analysis and processing functions are available at any magnification factor. The program supports any number of windows (images) simultaneously, limited only by available memory. Spatial calibration is available to provide real world dimensional measurements in units such as millimeters. Density or gray scale calibration is also available. ImageJ was designed with an open architecture that provides extensibility via Java plugins. Custom acquisition, analysis and processing plugins can be developed using ImageJ built in editor and Java compiler. User-written plugins make it possible to solve almost any image processing or analysis problem.

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Induced Mutant Resource

THIS RESOURCE IS NO LONGER IN SERVICE, documented on June 08, 2012. The function of the IMR is to select, import, cryopreserve, maintain, and distribute these important strains of mice to the research community. To improve their value for research, the IMR also undertakes genetic development of stocks, such as transferring mutant genes or transgenes to defined genetic backgrounds and combining transgenes and/or targeted mutations to create new mouse models for research. The function of the IMR is to: * select biomedically important stocks of transgenic, chemically induced, and targeted mutant mice * import these stocks into the Jackson Laboratory by rederivation procedures that rid them of any pathogens they might carry * cryopreserve embryos from these stocks to protect them against accidental loss and genetic contamination * backcross the mutation onto an inbred strain, if necessary * distribute them to the scientific community More than 1000 mutant stocks have been accepted by the IMR from 1992 through December 2006. Current holdings include models for research on cancer; breast cancer; immunological and inflammatory diseases; neurological diseases; behavioral, cardiovascular and heart diseases; developmental, metabolic and other diseases; reporter (e.g., GFP) and recombinase (e.g., cre/loxP) strains. About eight strains a month are being added to the IMR holdings. Research is being conducted on improved methods for assisted reproduction and speed congenic production. Most of the targeted mutants arrive on a mixed 129xC57BL/6 genetic background, and as many of these as possible are backcrossed onto an inbred strain (usually C57BL/6J). In addition, new mouse models are being created by intercrossing carriers of specific transgenes and/or targeted mutations. Simple sequence length polymorphism DNA markers are being used to characterize and evaluate differences between inbred strains, substrains, and embryonic stem cell lines.

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