Quantitative characterization of gene expression at single cell level combining reporter system with RT real-time PCR

ABSTRACTDirectly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatomThalassiosira pseudonanaas a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels inT. pseudonanaand demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

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Abstract 257: Clonal Analysis Of Multipotent Cardiovascular Progenitors That Generate Cardiomyocytes During Development

Circulation Research ◽

10.1161/res.115.suppl_1.257 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Konstantina Ioanna Sereti ◽

Paniz Kamran Rashani ◽

Peng Zhao ◽

Reza Ardehali

Keyword(s):

Single Cell ◽

Heart Development ◽

Cardiac Development ◽

Clonal Analysis ◽

Cardiac Cells ◽

Single Cell Level ◽

Reporter System ◽

Cell Level ◽

Cardiac Progenitors

It has been proposed that cardiac development in lower vertebrates is driven by the proliferation of cardiomyocytes. Similarly, cycling myocytes have been suggested to direct cardiac regeneration in neonatal mice after injury. Although, the role of cardiomyocyte proliferation in cardiac tissue generation during development has been well documented, the extent of this contribution as well as the role of other cell types, such as progenitor cells, still remains controversial. Here we used a novel stochastic four-color Cre-dependent reporter system (Rainbow) that allows labeling at a single cell level and retrospective analysis of the progeny. Cardiac progenitors expressing Mesp1 or Nkx2.5 were shown to be a source of cardiomyocytes during embryonic development while the onset of αMHC expression marked the developmental stage where the capacity of cardiac cells to proliferate diminishes significantly. Through direct clonal analysis we provide strong evidence supporting that cardiac progenitors, as opposed to mature cardiomyocytes, are the main source of cardiomyocytes during cardiac development. Moreover, we have identified quadri-, tri-, bi, and uni-potent progenitors that at a single cell level can generate cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Although existing cardiomyocytes undergo limited proliferation, our data indicates that it is mainly the progenitors that contribute to heart development. Furthermore, we show that the limited proliferation capacity of cardiomyocytes observed during normal development was enhanced following neonatal cardiac injury allowing almost complete regeneration of the scared tissue. However, this ability was largely absent in adult injured hearts. Detailed characterization of dividing cardiomyocytes and proliferating progenitors would greatly benefit the development of novel therapeutic options for cardiovascular diseases.

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Domain-Directed Polymerase Chain Reaction Capable of Distinguishing Bacterial from Host DNA at the Single-Cell Level: Characterization of a Systematic Method to Investigate Putative Bacterial Infection in Idiopathic Disease

Analytical Biochemistry ◽

10.1006/abio.1996.9896 ◽

1997 ◽

Vol 244 (2) ◽

pp. 328-339 ◽

Cited By ~ 21

Author(s):

C.R. Steinman ◽

B. Muralidhar ◽

G.J. Nuovo ◽

P.M. Rumore ◽

D. Yu ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Bacterial Infection ◽

Single Cell ◽

Single Cell Level ◽

Cell Level ◽

Chain Reaction ◽

Systematic Method ◽

Polymerase Chain ◽

Idiopathic Disease

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Gene expression profiling of the different stages of Arabidopsis thaliana trichome development on the single cell level

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2007.11.001 ◽

2008 ◽

Vol 46 (2) ◽

pp. 160-173 ◽

Cited By ~ 26

Author(s):

Sergiy Kryvych ◽

Victoria Nikiforova ◽

Michel Herzog ◽

Daniel Perazza ◽

Joachim Fisahn

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Single Cell ◽

Gene Expression Profiling ◽

Expression Profiling ◽

Single Cell Level ◽

Cell Level ◽

Trichome Development

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Isolation and characterization of individual functionally reactive cytotoxic T lymphocytes: conjugation, killing and recycling at the single cell level

European Journal of Immunology ◽

10.1002/eji.1830051205 ◽

1975 ◽

Vol 5 (12) ◽

pp. 818-822 ◽

Cited By ~ 130

Author(s):

D. Zagury ◽

Jacky Bernard ◽

Noelle Thierness ◽

M. Feldman ◽

G. Berke

Keyword(s):

T Lymphocytes ◽

Single Cell ◽

Cytotoxic T Lymphocytes ◽

Single Cell Level ◽

Cell Level ◽

Isolation And Characterization

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Unmasking cellular response of a bloom-forming alga to virus infection by resolving expression profiling at a single-cell level

10.1101/186981 ◽

2017 ◽

Author(s):

Shilo Rosenwasser ◽

Miguel J. Frada ◽

David Pilzer ◽

Ron Rotkopf ◽

Assaf Vardi

Keyword(s):

Gene Expression ◽

Viral Infection ◽

Single Cell ◽

Host Defense ◽

Host Response ◽

Expression Profiles ◽

Life Cycles ◽

Viral Gene ◽

Single Cell Level ◽

Cell Level

AbstractMarine viruses are major evolutionary and biogeochemical drivers of microbial life in the ocean. Host response to viral infection typically includes virus-induced rewiring of metabolic network to supply essential building blocks for viral assembly, as opposed to activation of anti-viral host defense. Nevertheless, there is a major bottleneck to accurately discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we use Emiliania huxleyi, a bloom-forming alga and its specific virus (EhV), as one of the most ecologically important host-virus model system in the ocean. Using automatic microfluidic setup to capture individual algal cells, we quantified host and virus gene expression on a single-cell resolution during the course of infection. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and uncover a yet unrecognized early phase in host response that occurs prior to viral expression. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and sensitive cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

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