RESUMO
The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human-secreted placental alkaline phosphatase (SEAP) and bacterial beta-lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.
Assuntos
Células PC12/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Vírus do Sarcoma Aviário/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Biomarcadores , Carbacol/farmacologia , Imunofluorescência , Expressão Gênica/fisiologia , Humanos , Células PC12/enzimologia , Ratos , Transcrição Gênica/fisiologia , Transfecção , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Vectors have been designed to optimise the expression of heterologous proteins in transfected mouse myeloma cells. The over-ridingly important DNA element contained in these constructs is the classical mouse immunoglobulin heavy chain enhancer. It is shown that even in the absence of a well-known promoter element, the enhancer can drive gene expression in stable cell transfectants and the main transcriptional start site utilized in such situations has been mapped to within the previously defined enhancer region. Using chicken lysozyme as a reporter function in these vectors, two transfected myeloma cell clones have been isolated which secrete this protein at levels 50-100-times as high as those usually obtained with the same vectors and it is shown that in molar terms this is at least as high as endogenous immunoglobulin produced by a related line. Analysis of these lines show that in one case only a single copy, and in the other two to three copies, of the apparently unrearranged vector have integrated at a single locus within the genome. Possible explanations for the high-level expression are discussed.
