Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo

1999 ◽  
Vol 17 (5) ◽  
pp. 557-561 ◽  
Author(s):  
Boris Hedtke ◽  
Martin Meixner ◽  
Sabine Gillandt ◽  
Ekkehard Richter ◽  
Thomas Börner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Rna Polymerases ◽  
Green Fluorescent

scholarly journals A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

2004 ◽  
Vol 15 (10) ◽  
pp. 4622-4632 ◽  
Author(s):  
Yasmina Bauer ◽  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Hanspeter Helfer ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Ashbya Gossypii ◽  
Growth Guidance ◽  
Characteristic Features ◽  
Green Fluorescent ◽  
Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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