scholarly journals In vivo bioluminescence for tracking cell fate and function

2011 ◽  
Vol 301 (3) ◽  
pp. H663-H671 ◽  
Author(s):  
Patricia E. de Almeida ◽  
Juliaan R. M. van Rappard ◽  
Joseph C. Wu
Keyword(s):  
Gene Expression ◽  
Cell Survival ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Cardiac Injury ◽  
Therapeutic Interventions ◽  
Luciferase Expression ◽  
Physiological Processes ◽  
And Function

Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.

scholarly journals Ultrasound Imaging of Gene Expression in Mammalian Cells

2019 ◽  
Author(s):  
Arash Farhadi ◽  
Gabrielle H. Ho ◽  
Daniel P. Sawyer ◽  
Raymond W. Bourdeau ◽  
Mikhail G. Shapiro
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Reporter Genes ◽  
Tissue Imaging ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Deep Tissue Imaging ◽  
And Function ◽  
Expression In Mammalian Cells

ABSTRACTThe study of cellular processes occurring inside intact organisms and the development of cell-based diagnostic and therapeutic agents requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling deep-tissue imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce the first mammalian acoustic reporter genes. Starting with an eleven-gene polycistronic gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of a unique class of intracellular air-filled protein nanostructures called gas vesicles. The scattering of ultrasound by these nanostructures allows mammalian cells to be visualized at volumetric densities below 0.5%, enables the monitoring of dynamic circuit-driven gene expression, and permits high-resolution imaging of gene expression in living animals. These mammalian acoustic reporter genes enable previously impossible approaches to monitoring the location, viability and function of mammalian cellsin vivo.

'Liver-type' 11β-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells

1994 ◽  
Vol 13 (2) ◽  
pp. 167-174 ◽  
Author(s):  
S C Low ◽  
K E Chapman ◽  
C R W Edwards ◽  
J R Seckl
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Mmtv Ltr

ABSTRACT 11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11β-HSD exist. One isoform (11β-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11β-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 β-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11β-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11β-HSD isoform. 11β-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11β-HSD1 activity in intact mammalian cells, and the possible role of 11β-HSD in regulating glucocorticoid access to GRs, we transfected rat 11β-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11β-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11β-HSD cDNA exhibited a dose-related increase in 11 β-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11β-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11β-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11β-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11β-HSD, but induced MMTV-LTR luciferase activity in the presence of 11β-HSD. These results indicate that rat 11β-HSD1 can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11β-HSD1 may regulate ligand access to GRs by reactivating inert glucocorticoids.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals Transcription modulation by CDK9 regulates inflammatory genes and RIPK3-MLKL-mediated necroptosis in periodontitis progression

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jiao Li ◽  
Jiahong Shi ◽  
Yue Pan ◽  
Yunhe Zhao ◽  
Fuhua Yan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Survival ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Acute Infection ◽  
Infection Process ◽  
Inhibitory Protein ◽  
Inflammatory Gene ◽  
Knock Down

AbstractCyclin-dependent kinase 9 (CDK9), one crucial molecule in promoting the transition from transcription pausing to elongation, is a critical modulator of cell survival and death. However, the pathological function of CDK9 in bacterial inflammatory diseases has never been explored. CDK9 inhibition or knock-down attenuated Porphyromonas gingivalis-triggered inflammatory gene expression. Gene-expression microarray analysis of monocytes revealed that knock-down of CDK9 not only affected inflammatory responses, but also impacted cell death network, especially the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-mediated necroptosis after P. gingivalis infection. Inhibition of CDK9 significantly decreased necroptosis with downregulation of both MLKL and phosphorylated MLKL. By regulating caspase-8 and cellular FLICE inhibitory protein (cFLIP), key molecules in regulating cell survival and death, CDK9 affected not only the classic RIPK1-RIPK3-mediated necroptosis, but also the alternate TIR-domain-containing adapter-inducing interferon-β-RIPK3-mediated necroptosis. CDK9 inhibition dampened pro-inflammatory gene production in the acute infection process in the subcutaneous chamber model in vivo. Moreover, CDK9 inhibition contributed to the decreased periodontal bone loss and inflammatory response induced by P. gingivalis in the periodontal micro-environment. In conclusion, by modulating the RIPK3-MLKL-mediated necroptosis, CDK9 inhibition provided a novel mechanism to impact the progress of bacterial infection in the periodontal milieu.

Multi-scale modeling of cell survival and death mediated by the p53 network: a systems pharmacology framework

2015 ◽  
Vol 11 (11) ◽  
pp. 3011-3021 ◽  
Author(s):  
Yuan Wang ◽  
Zihu Guo ◽  
Xuetong Chen ◽  
Wenjuan Zhang ◽  
Aiping Lu ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Fate ◽  
Mammalian Cells ◽  
Regulatory Process ◽  
Systems Pharmacology ◽  
Multi Scale ◽  
Scale Modeling ◽  
Multi Scale Modeling ◽  
P53 Network

The determination of cell fate is a key regulatory process for the development of complex organisms that are controlled by distinct genes in mammalian cells.

P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Julie Williams ◽  
Sanlin Robinson ◽  
Babak Alaei ◽  
Kimberly Homan ◽  
Maryam Clausen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Impact Analysis ◽  
Cell Types ◽  
Drug Uptake ◽  
Shear Stresses ◽  
The Matrix ◽  
And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

scholarly journals A-Kinase Anchoring Protein 1: Emerging Roles in Regulating Mitochondrial Form and Function in Health and Disease

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 298 ◽  
Author(s):  
Yujia Liu ◽  
Ronald A. Merrill ◽  
Stefan Strack
Keyword(s):  
Cell Survival ◽  
Intracellular Signaling ◽  
Neuronal Cell ◽  
Supporting Cell ◽  
In Vivo Studies ◽  
Form And Function ◽  
Anchoring Protein ◽  
Cancer Tumor ◽  
And Function

Best known as the powerhouse of the cell, mitochondria have many other important functions such as buffering intracellular calcium and reactive oxygen species levels, initiating apoptosis and supporting cell proliferation and survival. Mitochondria are also dynamic organelles that are constantly undergoing fission and fusion to meet specific functional needs. These processes and functions are regulated by intracellular signaling at the mitochondria. A-kinase anchoring protein 1 (AKAP1) is a scaffold protein that recruits protein kinase A (PKA), other signaling proteins, as well as RNA to the outer mitochondrial membrane. Hence, AKAP1 can be considered a mitochondrial signaling hub. In this review, we discuss what is currently known about AKAP1′s function in health and diseases. We focus on the recent literature on AKAP1′s roles in metabolic homeostasis, cancer and cardiovascular and neurodegenerative diseases. In healthy tissues, AKAP1 has been shown to be important for driving mitochondrial respiration during exercise and for mitochondrial DNA replication and quality control. Several recent in vivo studies using AKAP1 knockout mice have elucidated the role of AKAP1 in supporting cardiovascular, lung and neuronal cell survival in the stressful post-ischemic environment. In addition, we discuss the unique involvement of AKAP1 in cancer tumor growth, metastasis and resistance to chemotherapy. Collectively, the data indicate that AKAP1 promotes cell survival throug regulating mitochondrial form and function. Lastly, we discuss the potential of targeting of AKAP1 for therapy of various disorders.

A Comparative Study of Preparation Techniques for Improving the Viability of Striatal Grafts Using Vital Stains, in Vitro Cultures, and in Vivo Grafts

1996 ◽  
Vol 5 (6) ◽  
pp. 599-611 ◽  
Author(s):  
Rosemary A. Fricker ◽  
Roger A. Barker ◽  
James W. Fawcett ◽  
Stephen B. Dunnett
Keyword(s):  
Cell Survival ◽  
Cell Suspension ◽  
Single Cells ◽  
Rat Striatum ◽  
Adult Rat ◽  
Striatal Grafts ◽  
Vital Stains ◽  
And Function

Cell suspension grafts from embryonic striatal primordia placed into the adult rat striatum survive well and are able to alleviate a number of behavioral deficits caused by excitotoxic lesions to this structure. However, neither the anatomical connectivity between the graft and host nor the functional recovery elicited by the grafts is completely restored. One way in which the survival and function of embryonic striatal grafts may be enhanced is by the improvement of techniques for the preparation of the cell suspension prior to implantation, an issue that has been addressed only to a limited extent. We have evaluated a number of parameters during the preparation procedure, looking at the effects on cell survival over the first 24 h from preparation using vital dyes and the numbers of surviving neurons in vitro, after 4 days in culture, in addition to graft survival and function in vivo. Factors influencing cell survival include the type of trypsinization procedure and the age of donor tissues used for suspension preparation. The presence of DNase has no effect on cell viability but aids the dissociation of the tissue to form single cells. These results have important implications for the use of embryonic striatal grafts in animal models of Huntington's disease, and in any future clinical application of this research.

Surprising roles for phospholipid binding proteins revealed by high throughput geneticsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 565-574 ◽  
Author(s):  
Marissa A. LeBlanc ◽  
Christopher R. McMaster
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Essential Gene ◽  
Peer Review Process ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
And Function ◽  
Lipid Binding Proteins

Saccharomyces cerevisiae remains an ideal organism for studying the cell biological roles of lipids in vivo, as yeast has phospholipid metabolic pathways similar to mammalian cells, is easy and economical to manipulate, and is genetically tractable. The availability of isogenic strains containing specific genetic inactivation of each non-essential gene allowed for the development of a high-throughput method, called synthetic genetic analysis (SGA), to identify and describe precise pathways or functions associated with specific genes. This review describes the use of SGA to aid in elucidating the function of two lipid-binding proteins that regulate vesicular transport, Sec14 and Kes1. Sec14 was first identified as a phosphatidylcholine (PC) – phosphatidylinositol (PI) transfer protein required for viability, with reduced Sec14 function resulting in diminished vesicular transport out of the trans-Golgi. Although Sec14 is required for cell viability, inactivating the KES1 gene that encodes for a member of the oxysterol binding protein family in cells lacking Sec14 function results in restoration of vesicular transport and cell growth. SGA analysis identified a role for Kes1 and Sec14 in regulating the level and function of Golgi PI-4-phosphate (PI-4-P). SGA also determined that Sec14 not only regulates vesicular transport out of the trans-Golgi, but also transport from endosomes to the trans-Golgi. Comparing SGA screens in databases, coupled with genetic and cell biological analyses, further determined that the PI-4-P pool affected by Kes1 is generated by the PI 4-kinase Pik1. An important biological role for Sec14 and Kes1 revealed by SGA is coordinate regulation of the Pik1-generated Golgi PI-4-P pool that in turn is essential for vesicular transport into and out of the trans-Golgi.

Chromatin structure and function: the heretical path to an RNA transcription factor

1996 ◽  
Vol 74 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Margarida O. Krause
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Myc Gene ◽  
And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

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