Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA

Stéphane Esnault, James S. Malter

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV- driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t(1/2) in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t(1/2) of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded; suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM- CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow derailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.

Original languageEnglish (US)
Pages (from-to)5228-5234
Number of pages7
JournalJournal of Immunology
Volume163
Issue number10
StatePublished - Nov 15 1999

Fingerprint

Granulocyte-Macrophage Colony-Stimulating Factor
Eosinophils
Messenger RNA
Transfection
Terminal Repeat Sequences
Poisons
Regulator Genes
Green Fluorescent Proteins
Biological Assay
Northern Blotting
Complementary DNA
Enzyme-Linked Immunosorbent Assay
Cytokines
Technology
Genes
Proteins

ASJC Scopus subject areas

  • Immunology

Cite this

Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA. / Esnault, Stéphane; Malter, James S.

In: Journal of Immunology, Vol. 163, No. 10, 15.11.1999, p. 5228-5234.

Research output: Contribution to journalArticle

@article{1de77f2c8d89437891166eea891b31e3,
title = "Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA",
abstract = "Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2{\%} based on green fluorescent protein-positive cells. Promoter studies revealed CMV- driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t(1/2) in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t(1/2) of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded; suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM- CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow derailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.",
author = "St{\'e}phane Esnault and Malter, {James S.}",
year = "1999",
month = "11",
day = "15",
language = "English (US)",
volume = "163",
pages = "5228--5234",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "10",

}

TY - JOUR

T1 - Primary peripheral blood eosinophils rapidly degrade transfected granulocyte-macrophage colony-stimulating factor mRNA

AU - Esnault, Stéphane

AU - Malter, James S.

PY - 1999/11/15

Y1 - 1999/11/15

N2 - Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV- driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t(1/2) in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t(1/2) of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded; suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM- CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow derailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.

AB - Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMV- driven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t(1/2) in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t(1/2) of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded; suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM- CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow derailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.

UR - http://www.scopus.com/inward/record.url?scp=0033571755&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033571755&partnerID=8YFLogxK

M3 - Article

C2 - 10553043

AN - SCOPUS:0033571755

VL - 163

SP - 5228

EP - 5234

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 10

ER -