A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines.
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 non-macrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.
Beutler
B
B
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Brown
T
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D008070:Lipopolysaccharides; D009841:Oligonucleotides; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase