scholarly journals A Tendon-Specific Double Reporter Transgenic Mouse Enables Tracking Cell Lineage and Functions Alteration In Vitro and In Vivo

2021 ◽  
Vol 22 (20) ◽  
pp. 11189
Author(s):  
Rui Chen ◽  
Xunlei Zhou ◽  
Thomas Skutella
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Cell Lineage ◽  
Mouse Line ◽  
Reporter System ◽  
Specific Expression ◽  
Transgenic Mouse Line ◽  
Blue Fluorescent Protein

We generated and characterized a transgenic mouse line with the tendon-specific expression of a double fluorescent reporter system, which will fulfill an unmet need for animal models to support real-time monitoring cell behaviors during tendon development, growth, and repair in vitro and in vivo. The mScarlet red fluorescent protein is driven by the Scleraxis (Scx) promoter to report the cell lineage alteration. The blue fluorescent protein reporter is expressed under the control of the 3.6kb Collagen Type I Alpha 1 Chain (Col1a1) proximal promoter. In this promoter, the existence of two promoter regions named tendon-specific cis-acting elements (TSE1, TSE2) ensure the specific expression of blue fluorescent protein (BFP) in tendon tissue. Collagen I is a crucial marker for tendon regeneration that is a major component of healthy tendons. Thus, the alteration of function during tendon repair can be estimated by BFP expression. After mechanical stimulation, the expression of mScarlet and BFP increased in adipose-derived mesenchymal stem cells (ADMSCs) from our transgenic mouse line, and there was a rising trend on tendon key markers. These results suggest that our tendon-specific double reporter system is a novel model used to study cell re-differentiation and extracellular matrix alteration in vitro and in vivo.

scholarly journals A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽  
2021 ◽  
Vol 2 (3) ◽  
Author(s):  
Nelly Redolfi ◽  
Elisa Greotti ◽  
Giulia Zanetti ◽  
Tino Hochepied ◽  
Cristina Fasolato ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Brain Slices ◽  
Resonance Energy ◽  
Mouse Line ◽  
Isolated Cells ◽  
Genomic Locus ◽  
Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

Emergency prevention of extinction of a transgenic allele in a less-fertile transgenic mouse line by crossing with an inbred or outbred mouse strain coupled with assisted reproductive technologies

2007 ◽  
Vol 19 (8) ◽  
pp. 984 ◽  
Author(s):  
Anna Mayer ◽  
Diana Bulian ◽  
Hagen Scherb ◽  
Martin Hrabé de Angelis ◽  
Jörg Schmidt ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Low Fertility ◽  
In Vitro Fertilisation ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Green Fluorescent

Certain transgenic mouse lines are difficult to breed or archive and, consequently, their transgenes become lost. We examined a C57BL/6 mouse line (B6-tg), transgenic for green fluorescent protein (GFP) with low fertility, and its crosses with the more prolific inbred C3HeB/FeJ (C3) and outbred Swiss (SW) strains in order to assess the possibility of emergency prevention of extinction of a transgenic allele by using assisted reproductive technologies (ART). Out-crossing was performed by natural mating or in vitro fertilisation (IVF) with heterozygous mice. Most of the crossing combinations resulted in improved archiving and rederivation efficiencies of the transgenic allele. Natural crossing increased both mean litter size by two to three pups and the superovulatory rate from 69% for B6-tg to 70–90% for females from the out-crosses. Each plug-positive B6-tg female yielded an average of 4.6 two-cell embryos, whereas females from the out-crosses produced three- to fivefold that amount. After thawing, 13% of B6-tg embryos and 6–12% of out-crossed embryos developed into transgenic pups after transfer into recipients. After IVF with cryopreserved spermatozoa, cleavage rates were 4% for B6-tg, 22–37% for B6-tg oocytes out-crossed with C3 and SW spermatozoa, 9–49% for gametes from out-crossed mice and 28–44% for back-crosses with B6 oocytes. Transgenic pups were not derived from IVF with B6-tg gametes when either fresh or cryopreserved spermatozoa were used. Rederivation efficiencies were 7% and 4% from out-crosses of B6-tg oocytes with C3 and SW spermatozoa, respectively, 6–22% for gametes from out-crossed mice and 4–10% for the back-crosses. Although out-crossing changes the original genetic background, the strategy of crossing coupled with ART prevents the extinction of an allele of interest, especially where archiving and rederivation of the transgenic line fail.

Studies of intestinal morphology and cathepsin B expression in a transgenic mouse aiming at intestine-specific expression of Cath B-EGFP

2011 ◽  
Vol 392 (11) ◽  
Author(s):  
Maria Arampatzidou ◽  
Kristina Mayer ◽  
Maria E. Iolyeva ◽  
Seblewongel Gebre Asrat ◽  
Mirunalini Ravichandran ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Cathepsin B ◽  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Intestinal Morphology ◽  
Specific Expression ◽  
Transgenic Expression ◽  
Morphological Studies

AbstractCathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin Bin vivo. Previously we showed that the A33-antigen promoter could be successfully usedin vitroin order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chi-meric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translatedin situalthough the transgene was maintained over several generations.

scholarly journals A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129934 ◽  
Author(s):  
Stefanie Besser ◽  
Marit Sicker ◽  
Grit Marx ◽  
Ulrike Winkler ◽  
Volker Eulenburg ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Gabaergic Neurons ◽  
Mouse Line ◽  
Red Fluorescent Protein ◽  
Transgenic Mouse Line

scholarly journals Nas transgenic mouse line allows visualization of Notch pathway activity in vivo

genesis ◽  
2006 ◽  
Vol 44 (6) ◽  
pp. 277-286 ◽  
Author(s):  
Céline Souilhol ◽  
Sarah Cormier ◽  
Marie Monet ◽  
Sandrine Vandormael-Pournin ◽  
Anne Joutel ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Notch Pathway ◽  
Mouse Line ◽  
Pathway Activity ◽  
Transgenic Mouse Line

scholarly journals Transmission of tauopathy strains is independent of their isoform composition

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhuohao He ◽  
Jennifer D. McBride ◽  
Hong Xu ◽  
Lakshmi Changolkar ◽  
Soo-jung Kim ◽  
...  
Keyword(s):  
Human Brain ◽  
Transgenic Mouse ◽  
Mouse Line ◽  
Cell Type ◽  
Transgenic Mouse Line ◽  
Adult Human ◽  
Tau Isoforms ◽  
Underlying Mechanisms ◽  
Mouse Lines

AbstractThe deposition of pathological tau is a common feature in several neurodegenerative tauopathies. Although equal ratios of tau isoforms with 3 (3R) and 4 (4R) microtubule-binding repeats are expressed in the adult human brain, the pathological tau from different tauopathies have distinct isoform compositions and cell type specificities. The underlying mechanisms of tauopathies are unknown, partially due to the lack of proper models. Here, we generate a new transgenic mouse line expressing equal ratios of 3R and 4R human tau isoforms (6hTau mice). Intracerebral injections of distinct human tauopathy brain-derived tau strains into 6hTau mice recapitulate the deposition of pathological tau with distinct tau isoform compositions and cell type specificities as in human tauopathies. Moreover, through in vivo propagation of these tau strains among different mouse lines, we demonstrate that the transmission of distinct tau strains is independent of strain isoform compositions, but instead intrinsic to unique pathological conformations.

Non-invasive bioluminescence imaging of caspase-3 activity in Breast Cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14557-e14557
Author(s):  
C. C. Olsen ◽  
F. Li ◽  
Z. He ◽  
W. Li ◽  
C. Li
Keyword(s):  
Breast Cancer ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Fold Increase ◽  
Reporter System ◽  
Reporter Protein ◽  
Reporter Proteins

e14557 Background: Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase 3 activities, which are elevated at the execution phase of apoptosis. Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system.. During apoptosis, activated caspse 3 cleaves off the multi-ubiquitin domain from the reporter protein. This enable the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72hrs after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24 fold increase and fractionated RT (5Gy x3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminsecence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. No significant financial relationships to disclose.

scholarly journals Development of a transgenic mouse line for the evaluation of the androgen receptor activity in vivo

2003 ◽  
Vol 5 (S1) ◽  
Author(s):  
R Attar ◽  
C Cullinan ◽  
C-P Ho ◽  
M Swerdel ◽  
J Dell ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Transgenic Mouse ◽  
Receptor Activity ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Androgen Receptor Activity
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