Development ofEimeria nieschulzi(Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells

Thein vitroproduction of gametocytes and oocysts of the apicomplexan parasite genusEimeriais still a challenge in coccidiosis research. Until today, anin vitrodevelopment of gametocytes or oocysts had only been shown in someEimeriaspecies. For several mammalianEimeriaspecies, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitativein vitrodevelopment of the rat parasiteEimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selectedEimeria nieschulzistrain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-typeE. nieschulzistages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of anin vitrocultivation system forEimeriaspecies.

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The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽

10.3390/cancers13123024 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3024

Author(s):

Martin Fogtmann Berthelsen ◽

Maria Riedel ◽

Huiqiang Cai ◽

Søren H. Skaarup ◽

Aage K. O. Alstrup ◽

...

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Somatic Cells ◽

Genetic Alterations ◽

Lung Epithelial Cells ◽

Target Cells ◽

Multiple Gene ◽

Conditional Expression ◽

Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

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Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

Stem Cells International ◽

10.1155/2019/7281912 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

Thu T. Duong ◽

James Lim ◽

Vidyullatha Vasireddy ◽

Tyler Papp ◽

Hung Nguyen ◽

...

Keyword(s):

Cortical Neurons ◽

Transgene Expression ◽

Fluorescent Protein ◽

Ex Vivo ◽

Cell Types ◽

Egfp Expression ◽

Cell Type ◽

Egfp Gene ◽

Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

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Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00715.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. E807-E814 ◽

Cited By ~ 49

Author(s):

Lara R. Nyman ◽

Eric Ford ◽

Alvin C. Powers ◽

David W. Piston

Keyword(s):

Blood Flow ◽

Blood Glucose ◽

Pancreatic Islets ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Cell Types ◽

Β Cells ◽

Islet Blood Flow ◽

Significant Difference

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas.

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Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01152.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. H699-H707 ◽

Cited By ~ 46

Author(s):

Ellen Steward Pentz ◽

Maria Luisa S. Sequeira Lopez ◽

Magali Cordaillat ◽

R. Ariel Gomez

Keyword(s):

Chromatin Remodeling ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Histone H4 Acetylation ◽

Renin Gene ◽

Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

10.1101/2021.07.12.452139 ◽

2021 ◽

Author(s):

Giovanni Gallo ◽

Ioannis Mougiakos ◽

Mauricio Bianco ◽

Miriam Carbonaro ◽

Andrea Carpentieri ◽

...

Keyword(s):

Catalytic Mechanism ◽

Fluorescent Protein ◽

Efflux Pump ◽

Thermus Thermophilus ◽

Yellow Fluorescent Protein ◽

Arsenic Resistance ◽

Wide Range ◽

Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

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A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on “single-cell”-specific KO model of the distal nephron

AJP Renal Physiology ◽

10.1152/ajprenal.00286.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. F901-F906 ◽

Cited By ~ 22

Author(s):

Francesco Trepiccione ◽

Christelle Soukaseum ◽

Anna Iervolino ◽

Federica Petrillo ◽

Miriam Zacchia ◽

...

Keyword(s):

Single Cell ◽

Kidney Development ◽

Fluorescent Protein ◽

Collecting Duct ◽

Yellow Fluorescent Protein ◽

Cell Types ◽

Intercalated Cells ◽

Distal Nephron ◽

Principal Cells ◽

Different Cell Types

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

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The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽

10.1210/en.2004-1637 ◽

2005 ◽

Vol 146 (5) ◽

pp. 2336-2344 ◽

Cited By ~ 12

Author(s):

Masako Shimada ◽

Matthew J. Mahon ◽

Peter A. Greer ◽

Gino V. Segre

Keyword(s):

Parathyroid Hormone ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Small Subunit ◽

Hek293 Cells ◽

Calpain Inhibitor ◽

Dependent Manner ◽

Intact Cells ◽

Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

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113. DEVELOPMENT OF A METHOD OF SEMINIFEROUS TUBULE TRANSFECTION IN VITRO TO DEFINE POSTMEIOTIC GENE REGULATION

Reproduction Fertility and Development ◽

10.1071/srb09abs113 ◽

2009 ◽

Vol 21 (9) ◽

pp. 32

Author(s):

S. Danner ◽

C. Kirchhoff ◽

R. Ivell

Keyword(s):

Fluorescent Protein ◽

Transgenic Animals ◽

Cell Types ◽

Seminiferous Tubules ◽

Gene Promoters ◽

Cell Type ◽

Cell Type Specificity ◽

High Throughput Analysis ◽

Apparent Lack

Postmeiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. Here we report the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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