Isotopic assays for reporter gene activity.
This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol. In the assays described here, transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material using either thin-layer chromatography or phase-extraction. The second reporter system uses a kit to perform a simple two-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.
Kingston
R E
RE
Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Sheen
J
J
Moore
D
D
D002250:Carbon Radioisotopes; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000235:genetics; D002855:Chromatography, Thin Layer; D017930:Genes, Reporter; D019382:Human Growth Hormone / Q000032:analysis; D006801:Humans; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods*; D011863:Radioimmunoassay / Q000379:methods; D011868:Radioisotopes / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis*
D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D011868:Radioisotopes; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D015500:Chloramphenicol O-Acetyltransferase