R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Chen, You-Tzung; Tsai, Ming-Shian; Yang, Tsung‐Lin; Ku, Amy T.; Huang, Ke-Han; Huang, Cheng‐Yen; Chou, Fu-Ju; Fan, Hao; Hong, Jin‐Bon; Yen, Shuo-Ting; Wang, Wei-Le; Lin, Chang‐Ching; Hsu, Yu‐Chen; Su, Kang‐Yi; Su, I‐Chang; Jang, Chuan-Wei; Behringer, Richard R.; Favaro, Rebecca; Nicolis, Silvia K.; Chien, Chung‐Liang; Lin, Shu‐Wha; Yu, I-Shing

doi:10.1371/journal.pone.0046171

Cited by 12 publications

(9 citation statements)

References 52 publications

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“…Pax7+ SCs were labeled using the Pax7cre/ERT2 knock-in driver (Lepper et al, 2009) for efficient tamoxifen-inducible, Cre-mediated recombination and expression of indelible fluorescent lineage reporters: cytoplasmic eYFP (Srinivas et al, 2001) or chromatin associated H2B-GFP (Chen et al, 2012) (Figures S1A–S1C). The hind limb tibialis anterior (TA) muscle (Figure 1A), commonly used for studying regeneration, was viewed by two-photon IVM (Figure S1D).…”

Section: Resultsmentioning

confidence: 99%

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Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration

et al. 2016

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Summary How resident stem cells and their immediate progenitors rebuild tissues of pre-injury organization and size for proportional regeneration is not well understood. Using 3D, time-lapse intravital imaging for direct visualization of the muscle regeneration process in live mice, we report that extra-cellular matrix remnants from injured skeletal muscle fibers, “ghost fibers,” govern muscle stem/progenitor cell behaviors during proportional regeneration. Stem cells were immobile and quiescent without injury whereas their activated progenitors migrated and divided after injury. Unexpectedly, divisions and migration were primarily bi-directionally oriented along the ghost fiber longitudinal axis, allowing for spreading of progenitors throughout ghost fibers. Re-orienting ghost fibers impacted myogenic progenitors’ migratory paths and division planes, causing disorganization of regenerated muscle fibers. We conclude that ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury. This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.

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Section: Resultsmentioning

confidence: 99%

“…Both R26R eYFP (B6.129X1- Gt ( ROSA ) 26Sor tm1 ( EYFP) ) Cos /J) (Srinivas et al, 2001) and R26R GR (B6;129- Gt (ROSA)26Sor tm1Ytchn /J) (Chen et al, 2012) reporter mice were obtained from the Jackson Laboratory. Appropriate mating schemes were used to generate mice for SC lineage marking as described in the text.…”

Section: Methodsmentioning

confidence: 99%

Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration

et al. 2016

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“…developed a self-cleavable fusion protein comprised of H2B-EGFP and GPI-anchored mCherry (Chen et al, 2012). However, these fusion proteins are not compatible with many FRET biosensors that utilize CFP and YFP as the FRET pair, because precedent markers use the green-to-yellow fluorescence range.…”

Section: Discussionmentioning

confidence: 99%

“…Notably, a number of mouse reporter lines developed by Aizawa and his colleagues utilize the ROSA26 locus for ubiquitous expression and provide researchers the unique opportunity to visualize tissue architecture in vivo Shioi et al, 2011). Moreover, a T2A or P2A self-cleavable peptide can be used to connect the cell membrane marker and nucleus marker in a single polypeptide, facilitating equimolar expression of the two markers (Chen et al, 2012;Trichas et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

Imanishi

Murata

Sato

et al. 2018

Cell Struct. Funct.

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Abbreviations: H&E, hematoxylin and eosin; BAC, bacterial artificial chromosome; FRET, Förster resonance energy transfer; TPEM, two-photon excitation microscopy; FP, fluorescent protein; WPRE, woodchuck hepatitis virus (WHP) posttranscriptional regulatory element; DCGAN, deep convolutional generative adversarial networks. AbstractFor more than a century, hematoxylin and eosin (H&E) staining has been the de-facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in-vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.

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“…Another group has also recently established a R26R‐GR (GR; H2B‐EGFP‐2A‐mCherry‐GPI) knock‐in reporter mouse line (Chen et al . ).…”

Section: Visualization Of Subcellular Targetsmentioning

confidence: 97%

Reporter Mouse Lines for Fluorescence Imaging

2013

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The use of live imaging approaches to examine and understand the dynamic processes that take place during mouse development has become widespread. Several groups have reported their success in generating different reporter mouse lines that express a variety of fluorescent markers for imaging. However, there is currently no established database of the reporter mouse lines available for live imaging, such as the Cre transgenic lines (Cre-X-Mice). Researchers therefore often have difficulties in determining which reporter mouse line meets their research purposes. In this review, we summarize some of the reporter mouse lines that have been generated for live imaging studies, and discuss their characteristics.

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R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Cited by 12 publications

References 52 publications

Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration

Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

Reporter Mouse Lines for Fluorescence Imaging

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