Search results for "Gene Expression"

Messenger RNA Sequencing of Rare Cell Populations in the Lung and Lung-Draining Lymph Nodes

2017

Next-generation sequencing (NGS) techniques provide unique prospects for in-depth transcriptome analyses. Nevertheless, the emerging and still growing knowledge about the large diversity and heterogeneity of cells that participate in immunological responses in a tissue- and micromilieu-specific manner calls for advanced isolation and sequencing methods for the accurate quantification of gene expression in small cell populations and even individual cells from any organ or tissue. One of the major limitations in performing transcriptome analyses of rare cell populations was and still is quality and quantity of RNA that often limits analyses of complex mixtures of immune cell populations. Here…

Deciphering the Early Mouse Embryo Transcriptome by Low-Input RNA-Seq

2020

Early preimplantation embryos are precious and scarce samples that contain limited numbers of cells, which can be problematic for quantitative gene expression analyses. Nonetheless, low-input genome-wide techniques coupled with cDNA amplification steps have become a gold standard for RNA profiling of as minimal as a single blastomere. Here, we describe a single-cell/single-embryo RNA sequencing (RNA-seq) method, from embryo collection to sample validation steps prior to DNA library preparation and sequencing. Key quality controls and external Spike-In normalization approaches are also detailed.

PCF11 links alternative polyadenylation to formation and spontaneous regression of neuroblastoma

2018

AbstractDiversification at the transcriptome 3’end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. However the underlying mechanisms and functional consequences are poorly defined. Here, we identify extensive transcriptome-3’end-alterations in neuroblastoma, a tumour entity with a paucity of recurrent somatic mutations and an unusually high frequency of spontaneous regression. Utilising extensive RNAi-screening we reveal the landscape and drivers of transcriptome-3’end-diversification, discovering PCF11 as critical regulator, directing alternative polyadenylation (APA) of hundreds of transcripts including…

Sex-specific responses to cold in a very cold-tolerant, northern Drosophila species

2020

AbstractOrganisms can plastically alter resource allocation in response to changing environmental factors. For example, in harsh conditions organisms are expected to shift investment from reproduction towards survival, however, the factors and mechanisms that govern the magnitude of such shifts are relatively poorly studied. Here we compared the impact of cold on males and females of the highly cold-tolerant species Drosophila montana at the phenotypic and transcriptomic levels. Although both sexes showed similar changes in cold tolerance and gene expression in response to cold treatment, indicating that the majority of changes are concordant between the sexes, we identified a clear reducti…

2021

Physical activity is considered a promising preventive intervention to reduce the risk of developing Alzheimer’s disease (AD). However, the positive effect of therapeutic administration of physical activity has not been proven conclusively yet, likely due to confounding factors such as varying activity regimens and life or disease stages. To examine the impact of different routines of physical activity in the early disease stages, we subjected young 5xFAD and wild-type mice to 1-day (acute) and 30-day (chronic) voluntary wheel running and compared them with age-matched sedentary controls. We observed a significant increase in brain lactate levels in acutely trained 5xFAD mice relative to al…

Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors

2014

Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infe…

Production of the Sweet Protein Monellin in Transgetic Plants

1992

Monellin is a protein that elicits a flavor approximately 100,000 times sweeter than sugar on a molar basis. The protein exists naturally as a heterodimer, with its sweet flavor lost upon denaturation. A single–chain monellin gene, encoding both polypeptide chains linked by a hinge sequence, was placed under the control of constitutive and fruit–ripening specific promoters and transferred to lettuce and tomato. Expression of these genes in transgenic tomato and lettuce resulted in the accumulation of monellin protein in fruit and leaf, respectively, to significant levels. Production of monellin in transgenic fruits and vegetables represents an alternative strategy to enhance their flavor an…

Expression of a reporter gene is reduced by a ribozyme in transgenic plants

1994

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentration…

Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver.

2010

We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied i…

Homeostasis in the Central Dogma of molecular biology: the importance of mRNA instability

2019

Cell survival requires the control of biomolecule concentration, i.e. biomolecules should approach homeostasis. With information-carrying macromolecules, the particular concentration variation ranges depend on each type: DNA is not buffered, but mRNA and protein concentrations are homeostatically controlled, which leads to the ribostasis and proteostasis concepts. In recent years, we have studied the particular features of mRNA ribostasis and proteostasis in the model organism S. cerevisiae. Here we extend this study by comparing published data from three other model organisms: E. coli, S. pombe and cultured human cells. We describe how mRNA ribostasis is less strict than proteostasis. A co…