A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines

Bruce Beutler, Tracy Brown

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

We have prepared a construct (designated CATTNF) in which the mouse TNF (cachectin) coding sequence is replaced by a sequence encoding chloramphenicol acetyltransferase (CAT), with preservation of the TNF promoter and 3′-untranslated sequences known to be important in the regulation of gene expression. When activated by LPS, permanently transfected RAW 264.7 (mouse macrophage) cells synthesize large quantities of CAT. Unlike TNF itself, CAT is nonsecreted and quite stable in the macrophage cytoplasm. Fewer than 1,000 LPS-induced macrophages can easily be detected by CAT assay. Cells maintain the ability to respond to LPS in vivo; as such, when injected intravenously, they accurately report conditions required for the production of TNF in diverse tissues. These cells may thus be used for the detection of cachectin/TNF synthesis in mice under conditions in which endogenously produced cachectin/ TNF would be undetectable. Studies of the expression of CATTNF in nonmacrophage cell lines have revealed that the modified TNF gene is constitutively expressed in L-929 cells, but that its expression is tightly suppressed in HeLa cells and in NIH 3T3 cells. This finding would suggest that certain nonmacrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.

Original languageEnglish (US)
Pages (from-to)1336-1344
Number of pages9
JournalJournal of Clinical Investigation
Volume87
Issue number4
StatePublished - Apr 1991

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Chloramphenicol O-Acetyltransferase
Cell Line
Tumor Necrosis Factor-alpha
Genes
Macrophages
NIH 3T3 Cells
Gene Expression Regulation
HeLa Cells
Cytoplasm
Messenger RNA

Keywords

  • Chloramphenical acetyltransferase
  • Endotoxin
  • Gene regulation
  • Macrophage
  • Reporter construct
  • TNF

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines",
abstract = "We have prepared a construct (designated CATTNF) in which the mouse TNF (cachectin) coding sequence is replaced by a sequence encoding chloramphenicol acetyltransferase (CAT), with preservation of the TNF promoter and 3′-untranslated sequences known to be important in the regulation of gene expression. When activated by LPS, permanently transfected RAW 264.7 (mouse macrophage) cells synthesize large quantities of CAT. Unlike TNF itself, CAT is nonsecreted and quite stable in the macrophage cytoplasm. Fewer than 1,000 LPS-induced macrophages can easily be detected by CAT assay. Cells maintain the ability to respond to LPS in vivo; as such, when injected intravenously, they accurately report conditions required for the production of TNF in diverse tissues. These cells may thus be used for the detection of cachectin/TNF synthesis in mice under conditions in which endogenously produced cachectin/ TNF would be undetectable. Studies of the expression of CATTNF in nonmacrophage cell lines have revealed that the modified TNF gene is constitutively expressed in L-929 cells, but that its expression is tightly suppressed in HeLa cells and in NIH 3T3 cells. This finding would suggest that certain nonmacrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.",
keywords = "Chloramphenical acetyltransferase, Endotoxin, Gene regulation, Macrophage, Reporter construct, TNF",
author = "Bruce Beutler and Tracy Brown",
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AU - Brown, Tracy

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N2 - We have prepared a construct (designated CATTNF) in which the mouse TNF (cachectin) coding sequence is replaced by a sequence encoding chloramphenicol acetyltransferase (CAT), with preservation of the TNF promoter and 3′-untranslated sequences known to be important in the regulation of gene expression. When activated by LPS, permanently transfected RAW 264.7 (mouse macrophage) cells synthesize large quantities of CAT. Unlike TNF itself, CAT is nonsecreted and quite stable in the macrophage cytoplasm. Fewer than 1,000 LPS-induced macrophages can easily be detected by CAT assay. Cells maintain the ability to respond to LPS in vivo; as such, when injected intravenously, they accurately report conditions required for the production of TNF in diverse tissues. These cells may thus be used for the detection of cachectin/TNF synthesis in mice under conditions in which endogenously produced cachectin/ TNF would be undetectable. Studies of the expression of CATTNF in nonmacrophage cell lines have revealed that the modified TNF gene is constitutively expressed in L-929 cells, but that its expression is tightly suppressed in HeLa cells and in NIH 3T3 cells. This finding would suggest that certain nonmacrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.

AB - We have prepared a construct (designated CATTNF) in which the mouse TNF (cachectin) coding sequence is replaced by a sequence encoding chloramphenicol acetyltransferase (CAT), with preservation of the TNF promoter and 3′-untranslated sequences known to be important in the regulation of gene expression. When activated by LPS, permanently transfected RAW 264.7 (mouse macrophage) cells synthesize large quantities of CAT. Unlike TNF itself, CAT is nonsecreted and quite stable in the macrophage cytoplasm. Fewer than 1,000 LPS-induced macrophages can easily be detected by CAT assay. Cells maintain the ability to respond to LPS in vivo; as such, when injected intravenously, they accurately report conditions required for the production of TNF in diverse tissues. These cells may thus be used for the detection of cachectin/TNF synthesis in mice under conditions in which endogenously produced cachectin/ TNF would be undetectable. Studies of the expression of CATTNF in nonmacrophage cell lines have revealed that the modified TNF gene is constitutively expressed in L-929 cells, but that its expression is tightly suppressed in HeLa cells and in NIH 3T3 cells. This finding would suggest that certain nonmacrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.

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