scholarly journals Gene Profiles and Electrophysiology of Doublecortin-Expressing Cells in the Subventricular Zone after Ischemic Stroke

2008 ◽  
Vol 29 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Xian Shuang Liu ◽  
Michael Chopp ◽  
Xue Guo Zhang ◽  
Rui Lan Zhang ◽  
Ben Buller ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Subventricular Zone ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
Brain Slices ◽  
Resting Membrane Potential ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Neuronal Marker ◽  
Gene Profiles

Stroke increases neuroblasts in the subventricular zone (SVZ) of the lateral ventricle and these neuroblasts migrate toward the ischemic boundary to replace damaged neurons. Using brain slices from the nonischemic adult rat and transgenic mice that expressed enhanced green fluorescent protein (EGFP) concomitantly with doublecortin (DCX), a marker for migrating neuroblasts, we recorded electrophysiological characteristics while simultaneously analyzing the gene expression in single SVZ cells. We found that SVZ cells expressing the DCX gene from the nonischemic rat had a mean resting membrane potential (RMP) of −30 mV. DCX—EGFP-positive cells in the nonischemic SVZ of the transgenic mouse had a mean RMP of −25 ± 7 mV and did not exhibit Na+ currents, characteristic of immature neurons. However, DCX—EGFP-positive cells in the ischemic SVZ exhibited a hyperpolarized mean RMP of −54 ± 18 mV and displayed Na+ currents, indicative of more mature neurons. Single-cell multiplex RT-PCR analysis revealed that DCX—EGFP-positive cells in the nonischemic SVZ of the transgenic mouse expressed high neural progenitor marker genes, Sox2 and nestin, but not mature neuronal marker genes. In contrast, DCX—EGFP-positive cells in the ischemic SVZ expressed tyrosine hydroxylase, a mature neuronal marker gene. Together, these data indicate that stroke changes gene profiles and the electrophysiology of migrating neuroblasts.

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+]).

scholarly journals Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Nele Tamberg ◽  
Valeria Lulla ◽  
Rennos Fragkoudis ◽  
Aleksei Lulla ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Marker Gene ◽  
Virus Production ◽  
Marker Genes ◽  
Replicase Gene ◽  
Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5223-5232 ◽  
Author(s):  
Naoshi Obara ◽  
Norio Suzuki ◽  
Kibom Kim ◽  
Toshiro Nagasawa ◽  
Shigehiko Imagawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Marker Genes ◽  
Central Vein ◽  
Specific Expression ◽  
Neuronal Marker ◽  
Nucleotide Mutation ◽  
Gata Box

Abstract In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type–specific expression of the Epo gene.

scholarly journals Development of plant regeneration and Agrobacterium tumefaciens-mediated transformation methodology for Physalis pruinosa

2018 ◽  
Author(s):  
Kerry Swartwood ◽  
Joyce Van Eck
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Genetic Engineering ◽  
Large Scale ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Scale Production ◽  
Hypocotyl Explants

AbstractPhysalis pruinosa, also known as groundcherry, produces a small, yellow, highly nutritious edible fruit that is enveloped by a papery husk. In order for the potential of large-scale production of P. pruinosa fruit to be realized, undesirable characteristics, such as an unmanageable, sprawling growth habit and extensive fruit drop, need to be improved by exploiting approaches available through plant breeding, genetic engineering, and gene editing. In this study, we established plant regeneration and Agrobacterium tumefaciens-mediated methods to allow application of genetic engineering and gene editing of P. pruinosa. Cotyledon and hypocotyl explants from 7 – 8-day-old in vitro-grown seedlings were assessed for plant regeneration. Explants were cultured for 2 weeks on a Murashige and Skoog salts-based medium that contained 2 mg/L zeatin followed by transfer to medium containing 1 mg/L zeatin. Only hypocotyl explants regenerated shoots. Hypocotyl explants were infected with Agrobacterium tumefaciens strain AGL1 containing the pJL33 binary vector that has the green fluorescent protein (GFP) reporter and neomycin phosphotransferase II (nptII) selectable marker genes. After cocultivation, explants were cultured on selective plant regeneration medium that contained 50, 100, 200, 250, and 300 mg/L kanamycin to determine the most effective level for efficient recovery of transgenic lines. Based on rooting of regenerated shoots on selective medium, GFP visualization, and PCR analysis for the presence of the nptII gene, medium containing 200 mg/L kanamycin resulted in the highest transformation efficiency at 24%. This study sets the foundation for future genetic engineering and gene editing approaches for improvement of P. pruinosa.

scholarly journals Patterns and Dynamics of Subventricular Zone Neuroblast Migration in the Ischemic Striatum of the Adult Mouse

2009 ◽  
Vol 29 (7) ◽  
pp. 1240-1250 ◽  
Author(s):  
Rui L Zhang ◽  
Michael Chopp ◽  
Sara R Gregg ◽  
Yier Toh ◽  
Cindi Roberts ◽  
...  
Keyword(s):  
Subventricular Zone ◽  
Adult Mouse ◽  
Fluorescent Protein ◽  
Brain Slices ◽  
Time Lapse ◽  
Adult Mouse Brain ◽  
Neuroblast Migration ◽  
Multiple Processes ◽  
Migratory Routes ◽  
Green Fluorescent

The migratory behavior of neuroblasts after a stroke is poorly understood. Using time-lapse microscopy, we imaged migration of neuroblasts and cerebral vessels in living brain slices of adult doublecortin (DCX, a marker of neuroblasts) enhanced green fluorescent protein (eGFP) transgenic mice that were subjected to 7 days of stroke. Our results show that neuroblasts originating in the subventricular zone (SVZ) of adult mouse brain laterally migrated in chains or individually to reach the ischemic striatum. The chains were initially formed at the border between the SVZ and the striatum by neuroblasts in the SVZ and then extended to the striatum. The average speed of DCX-eGFP-expressing cells within chains was 28.67 ± 1.04 μm/h, which was significantly faster ( P < 0.01) than the speed of the cells in the SVZ (17.98 ± 0.57 μm/h). Within the ischemic striatum, individual neuroblasts actively extended or retracted their processes, suggestive of probing the immediate microenvironment. The neuroblasts close to cerebral blood vessels exhibited multiple processes. Our data suggest that neuroblasts actively interact with the microenvironment to reach the ischemic striatum by multiple migratory routes.

The Aedes aegypti (Diptera: Culicidae) hsp83 Gene Promoter Drives Strong Ubiquitous DsRed and ZsGreen Marker Expression in Transgenic Mosquitoes

2021 ◽  
Author(s):  
Sophia H Webster ◽  
Maxwell J Scott
Keyword(s):  
Aedes Aegypti ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Marker Gene ◽  
Gene Promoter ◽  
Green Fluorescent Proteins ◽  
Marker Genes ◽  
Green Fluorescent ◽  
Population Suppression ◽  
Transgenic Mosquitoes

Abstract Transgenic strains of the mosquito disease vector Aedes aegypti (L.) are being developed for population suppression or modification. Transgenic mosquitoes are identified using fluorescent protein genes. Here we describe DsRed and ZsGreen marker genes driven by the constitutive Ae. aegypti heat shock protein 83 (hsp83) promoter in transgenic mosquitoes. Transgenic larvae and pupae show strong full body expression of the red and green fluorescent proteins. This greatly assists in screening for transgenic individuals while making new or maintaining already established lines. Transient marker gene expression after embryo microinjection was readily visible in developing larvae allowing the separation of individuals that are more likely to produce transgenic offspring. The strongly expressed marker genes developed in this study should facilitate the detection of transgenic Ae. aegypti larvae or pupae in the field.

scholarly journals Spatial Maps of Hepatocellular Carcinoma Transcriptomes Highlight an Unexplored Landscape of Heterogeneity and a Novel Gene Signature for Survival

2021 ◽  
Author(s):  
Nan Zhao ◽  
Yanhui Zhang ◽  
Runfen Cheng ◽  
Danfang Zhang ◽  
Fan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Drug Targets ◽  
Marker Gene ◽  
Risk Groups ◽  
Gene Signature ◽  
Low Risk ◽  
Marker Genes ◽  
Specific Marker ◽  
Gene Profiles

Abstract Background: Hepatocellular carcinoma (HCC) are often present with satellite nodules, rendering current curative treatments ineffective in many patients. The heterogeneity of HCC is a major challenge in personalized medicine. The emergence of spatial transcriptomics (ST) provides a powerful strategy for delineating the complex molecular landscapes of tumors. Methods: In this study, we investigated tissue-wide gene expression heterogeneity in tumor and adjacent nonneoplastic tissues using ST technology. We analyzed the transcriptomes of nearly 10820 tissue regions and identified main gene expression clusters and their specific marker genes (differentially expressed genes, DEGs) in patients. The DEGs were analyzed from two perspectives. First of all, we identified two distinct gene profiles associated with satellite nodules and conducted a more comprehensive analysis for both gene profiles. Their clinical relevance for human HCC was validated with KM Plotter. Secondly, we screened DEGs with TCGA database to divide the HCC cohort into high- and low-risk groups according to Cox analysis. HCC patients from the ICGC cohort were used for validation. Kaplan Meier analysis was used to compare the overall survival (OS) between high- and low-risk groups. Univariate and multivariate Cox analyses were applied to determine the independent predictors for OS. Results: Novel markers for the prediction of satellite nodules and a tumor clusters specific marker genes signature model(6 genes) for HCC prognosis was constructed, respectively. Conclusion: The establishment of marker gene profiles may be an important step towards an unbiased view of HCC and the 6-genes signature can be used for prognostic prediction in HCC. This analysis will help us to clarify one of the possible soucres of HCC heterogeneity, uncover pathogenic mechanisms and novel anti-tumor drug targets.

scholarly journals Oxytocin Modulation of Maternal Behavior and Its Association With Immunological Activity in Rats With Cesarean Delivery

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110147
Author(s):  
Tong Li ◽  
Shu-Wei Jia ◽  
Dan Hou ◽  
Xiaoran Wang ◽  
Dongyang Li ◽  
...  
Keyword(s):  
Cesarean Delivery ◽  
Maternal Behavior ◽  
Inflammatory Cytokine ◽  
Adrenocorticotropic Hormone ◽  
Brain Slices ◽  
Relative Weight ◽  
Resting Membrane Potential ◽  
Cytokine Release ◽  
Immunological Activity ◽  
Fos Expression

Oxytocin (OT), a neuropeptide produced in the supraoptic (SON) and paraventricular (PVN) nuclei, is not only essential for lactation and maternal behavior but also for normal immunological activity. However, mechanisms underlying OT regulation of maternal behavior and its association with immunity around parturition, particularly under mental and physical stress, remain unclear. Here, we observed effects of OT on maternal behavior in association with immunological activity in rats after cesarean delivery (CD), a model of reproductive stress. CD significantly reduced maternal interests to the pups throughout postpartum day 1-8. On postpartum day 5, CD decreased plasma OT levels and thymic index but increased vasopressin, interleukin (IL)-1β, IL-6 and IL-10 levels. CD had no significant effect on plasma adrenocorticotropic hormone and corticosterone levels. In the hypothalamus, CD decreased corticotropin-releasing hormone contents in the PVN but increased OT contents in the PVN and SON and OT release from hypothalamic implants. CD also increased c-Fos expression, particularly in the cytoplasm of OT neurons. Lastly, CD depolarized resting membrane potential and increased spike width while increasing the variability of the firing rate of OT neurons in brain slices. Thus, CD can increase hypothalamic OT contents and release but reduce pituitary release of OT into the blood, which is associated with depressive-like maternal behavior, increased inflammatory cytokine release and decreased relative weight of the thymus.

scholarly journals Multi-view feature selection for identifying gene markers: a diversified biological data driven approach

2020 ◽  
Vol 21 (S18) ◽  
Author(s):  
Sudipta Acharya ◽  
Laizhong Cui ◽  
Yi Pan
Keyword(s):  
Gene Expression ◽  
Feature Selection ◽  
Gene Selection ◽  
Marker Gene ◽  
Biological Data ◽  
Protein Interaction Data ◽  
Marker Genes ◽  
Data Sets ◽  
Gene Markers ◽  
Multi Objective

Abstract Background In recent years, to investigate challenging bioinformatics problems, the utilization of multiple genomic and proteomic sources has become immensely popular among researchers. One such issue is feature or gene selection and identifying relevant and non-redundant marker genes from high dimensional gene expression data sets. In that context, designing an efficient feature selection algorithm exploiting knowledge from multiple potential biological resources may be an effective way to understand the spectrum of cancer or other diseases with applications in specific epidemiology for a particular population. Results In the current article, we design the feature selection and marker gene detection as a multi-view multi-objective clustering problem. Regarding that, we propose an Unsupervised Multi-View Multi-Objective clustering-based gene selection approach called UMVMO-select. Three important resources of biological data (gene ontology, protein interaction data, protein sequence) along with gene expression values are collectively utilized to design two different views. UMVMO-select aims to reduce gene space without/minimally compromising the sample classification efficiency and determines relevant and non-redundant gene markers from three cancer gene expression benchmark data sets. Conclusion A thorough comparative analysis has been performed with five clustering and nine existing feature selection methods with respect to several internal and external validity metrics. Obtained results reveal the supremacy of the proposed method. Reported results are also validated through a proper biological significance test and heatmap plotting.

PPIT: an R package for inferring microbial taxonomy from nifH sequences

2021 ◽  
Author(s):  
Bennett J Kapili ◽  
Anne E Dekas
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Query Sequence ◽  
Marker Gene ◽  
R Package ◽  
Supplementary Information ◽  
Marker Genes ◽  
Pairwise Identity ◽  
Metabolic Marker ◽  
Microbial Taxonomy

Abstract Motivation Linking microbial community members to their ecological functions is a central goal of environmental microbiology. When assigned taxonomy, amplicon sequences of metabolic marker genes can suggest such links, thereby offering an overview of the phylogenetic structure underpinning particular ecosystem functions. However, inferring microbial taxonomy from metabolic marker gene sequences remains a challenge, particularly for the frequently sequenced nitrogen fixation marker gene, nitrogenase reductase (nifH). Horizontal gene transfer in recent nifH evolutionary history can confound taxonomic inferences drawn from the pairwise identity methods used in existing software. Other methods for inferring taxonomy are not standardized and require manual inspection that is difficult to scale. Results We present Phylogenetic Placement for Inferring Taxonomy (PPIT), an R package that infers microbial taxonomy from nifH amplicons using both phylogenetic and sequence identity approaches. After users place query sequences on a reference nifH gene tree provided by PPIT (n = 6317 full-length nifH sequences), PPIT searches the phylogenetic neighborhood of each query sequence and attempts to infer microbial taxonomy. An inference is drawn only if references in the phylogenetic neighborhood are: (1) taxonomically consistent and (2) share sufficient pairwise identity with the query, thereby avoiding erroneous inferences due to known horizontal gene transfer events. We find that PPIT returns a higher proportion of correct taxonomic inferences than BLAST-based approaches at the cost of fewer total inferences. We demonstrate PPIT on deep-sea sediment and find that Deltaproteobacteria are the most abundant potential diazotrophs. Using this dataset we show that emending PPIT inferences based on visual inspection of query sequence placement can achieve taxonomic inferences for nearly all sequences in a query set. We additionally discuss how users can apply PPIT to the analysis of other marker genes. Availability PPIT is freely available to non-commercial users at https://github.com/bkapili/ppit. Installation includes a vignette that demonstrates package use and reproduces the nifH amplicon analysis discussed here. The raw nifH amplicon sequence data have been deposited in the GenBank, EMBL, and DDBJ databases under BioProject number PRJEB37167. Supplementary information Supplementary data are available at Bioinformatics online.

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