scholarly journals Functional Characterization of Genetically Labeled Gonadotropes

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 2701-2711 ◽  
Author(s):  
Shuping Wen ◽  
Jürgen R. Schwarz ◽  
Dragos Niculescu ◽  
Crenguta Dinu ◽  
Christiane K. Bauer ◽  
...  
Keyword(s):  
Mouse Model ◽  
Anterior Pituitary ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Functional Characterization ◽  
Embryonic Stem ◽  
Significant Heterogeneity ◽  
Protein Fluorescence ◽  
Electrophysiological Responses ◽  
Cre Recombination

Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.

scholarly journals Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory

2019 ◽  
Vol 10 ◽  
Author(s):  
Mélanie J.A. Body ◽  
Dhruveesh F. Dave ◽  
Clayton M. Coffman ◽  
Taylor Y. Paret ◽  
Abraham J. Koo ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Insect Herbivory ◽  
Protein Fluorescence

Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis

Blood ◽  
2011 ◽  
Vol 117 (13) ◽  
pp. 3521-3528 ◽  
Author(s):  
David Hills ◽  
Ruby Gribi ◽  
Jan Ure ◽  
Natalija Buza-Vidas ◽  
Sidinh Luc ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hox Genes ◽  
Fetal Liver ◽  
Yellow Fluorescent Protein ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Hematopoietic Stem ◽  
Reporter Mouse

Abstract Hoxb4 overexpression promotes dramatic expansion of bone marrow (BM) hematopoietic stem cells (HSCs) without leukemic transformation and induces development of definitive HSCs from early embryonic yolk sac and differentiating embryonic stem cells. Knockout studies of Hoxb4 showed little effect on hematopoiesis, but interpretation of these results is obscured by the lack of direct evidence that Hoxb4 is expressed in HSCs and possible compensatory effects of other (Hox) genes. To evaluate accurately the pattern of Hoxb4 expression and to gain a better understanding of the physiologic role of Hoxb4 in the hemato-poietic system, we generated a knock-in Hoxb4–yellow fluorescent protein (YFP) reporter mouse model. We show that BM Lin−Sca1+c-Kit+ cells express Hoxb4-YFP and demonstrate functionally in the long-term repopulation assay that definitive HSCs express Hoxb4. Similarly, aorta-gonad-mesonephrous–derived CD45+CD144+ cells, enriched for HSCs, express Hoxb4. Furthermore, yolk sac and placental HSC populations express Hoxb4. Unexpectedly, Hoxb4 expression in the fetal liver HSCs is lower than in the BM, reaching negligible levels in some HSCs, suggesting an insignificant role of Hoxb4 in expansion of fetal liver HSCs. Hoxb4 expression therefore would not appear to correlate with the cycling status of fetal liver HSCs, although highly proliferative HSCs from young BM show strong Hoxb4 expression.

scholarly journals Ascl1 Lineage Cells Contribute to Ischemia-Induced Neurogenesis and Oligodendrogenesis

2010 ◽  
Vol 31 (2) ◽  
pp. 614-625 ◽  
Author(s):  
Rui Lan Zhang ◽  
Michael Chopp ◽  
Cindi Roberts ◽  
Longfei Jia ◽  
Min Wei ◽  
...  
Keyword(s):  
Corpus Callosum ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Adult Brain ◽  
Oligodendrocyte Progenitor Cells ◽  
Ischemic Brain ◽  
Helix Loop Helix ◽  
Cre Recombination ◽  
Cell Phenotypes

Neural and oligodendrocyte progenitor cells in the adult brain express Ascl1 (also known as Mash1), a basic helix-loop-helix transcription factor. We examined the progeny and fate of this progenitor population in adult male Ascl1-CreER™R26R-stop-yellow fluorescent protein mice subjected to right middle cerebral occlusion over 60 days after stroke using inducible Cre recombination to label Ascl1-expressing cells at poststroke days 2 to 6 in vivo. Seven days after stroke, a substantial increase in Ascl1 lineage cells was detected in the ipsilateral subventricular zone (SVZ), striatum, and corpus callosum. These cells exhibited proliferating progenitor cell phenotypes (Sox2+, BrdU+, and Ki67+). Although Ascl1 lineage cells in the ipsilateral SVZ gradually decreased during 14 to 60 days after stroke, Ascl1 lineage cells in the ischemic striatum revealed a remarkable increase during this period. Thirty and sixty days after stroke, Ascl1 lineage cells in the ischemic striatum gave rise to GABAergic neurons and mature oligodendrocytes. In contrast, none of the Ascl1 lineage cells in the contralateral striatum exhibited neuronal and oligodendrocyte phenotypes. Moreover, Ascl1 lineage cells in the corpus callosum were only fated to become mature oligodendrocytes. Our data suggest that Ascl1 lineage cells contribute to stroke-induced neurogenesis and oligodendrogenesis in the adult ischemic brain.

scholarly journals A mouse model of yellow fluorescent protein (YFP) expression in hematopoietic cells to assess leukocyte–endothelial interactions in the microcirculation

2009 ◽  
Vol 78 (3) ◽  
pp. 294-300 ◽  
Author(s):  
Thomas Kampfrath ◽  
Jeffrey A. Deiuliis ◽  
Susan D. Moffatt-Bruce ◽  
Jeffrey Anderson ◽  
Qinghua Sun ◽  
...  
Keyword(s):  
Mouse Model ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Hematopoietic Cells

scholarly journals Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Alenka Hrovat ◽  
Apolonija Zavec ◽  
Azra Pogačnik ◽  
Robert Frangež ◽  
Milka Vrecl
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Functional Characterization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enhanced Yellow Fluorescent Protein ◽  
Hek 293 ◽  
Transfected Cell Line

AbstractThis study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

2007 ◽  
Vol 12 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Mamatha Sauermann ◽  
Florian Hahne ◽  
Christian Schmidt ◽  
Meher Majety ◽  
Heiko Rosenfelder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Specific Antibody ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Fragmentation ◽  
Protein Fluorescence ◽  
Transfected Cells ◽  
High Throughput Assay

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry—based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. ( Journal of Biomolecular Screening 2007:510-520)

scholarly journals Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells

2014 ◽  
Vol 25 (13) ◽  
pp. 1958-1968 ◽  
Author(s):  
Kathleen Lisaingo ◽  
Evert-Jan Uringa ◽  
Peter M. Lansdorp
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Genome Instability ◽  
Yellow Fluorescent Protein ◽  
Embryonic Stem ◽  
Specific Protein ◽  
Protein Cleavage ◽  
Telomeric Dna ◽  
Cellular Processes ◽  
Telomere Protein

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.

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