1. Developmental Biology
Download icon

In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

  1. Li He  Is a corresponding author
  2. Richard Binari
  3. Jiuhong Huang
  4. Julia Falo-Sanjuan
  5. Norbert Perrimon  Is a corresponding author
  1. Harvard Medical School, United States
  2. Chongqing University of Arts and Sciences, China
  3. Tufts University, United States
Tools and Resources
  • Cited 5
  • Views 6,125
  • Annotations
Cite this article as: eLife 2019;8:e46181 doi: 10.7554/eLife.46181

Abstract

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

Article and author information

Author details

  1. Li He

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    Li_He@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2155-606X
  2. Richard Binari

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jiuhong Huang

    International Academy of Targeted Therapeutics and Innovation, Chongqing University of Arts and Sciences, Chongqing, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Julia Falo-Sanjuan

    School of Graduate Biomedical Sciences, Tufts University, Medford, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    For correspondence
    perrimon@receptor.med.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X

Funding

National Institute of General Medical Sciences

  • Norbert Perrimon

Damon Runyon Cancer Research Foundation

  • Li He

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Hugo J Bellen, Baylor College of Medicine, United States

Publication history

  1. Received: February 22, 2019
  2. Accepted: May 28, 2019
  3. Accepted Manuscript published: May 29, 2019 (version 1)
  4. Version of Record published: July 26, 2019 (version 2)

Copyright

© 2019, He et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,125
    Page views
  • 1,245
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Developmental Biology
    Noriko Ichino et al.
    Tools and Resources

    One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1,200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

    1. Cell Biology
    2. Developmental Biology
    Guillermo Marques et al.
    Feature Article

    A variety of microscopy techniques are used by researchers in the life and biomedical sciences. As these techniques become more powerful and more complex, it is vital that scientific articles containing images obtained with advanced microscopes include full details about how each image was obtained. To explore the reporting of such details we examined 240 original research articles published in eight journals. We found that the quality of reporting was poor, with some articles containing no information about how images were obtained, and many articles lacking important basic details. Efforts by researchers, funding agencies, journals, equipment manufacturers and staff at shared imaging facilities are required to improve the reporting of experiments that rely on microscopy techniques.