Production of human granulocyte macrophage colony stimulating factor using goat beta-casein promoter in the milk of transgenic mice

Abstract

Human granulocyte macrophage-colony stimulating factor (GM-CSF) is the most widely used human hemopoietic factors to remedy different forms of neutropenia, chemotherapy-induced leucopenia, and in the mobilization of progenitor cells for autologuous or allogenic transplantation. This colony stimulating factor maintains the level of blood cells within narrow limits, regulate and coordinate the proliferative activity of hemopoietic tissue, and play an essential role in response to emergent situations; for example, blood loss, or acute infection, and so on. Although these factors have been produced by many production technologies, recombinant human colony stimulating factors expressed using E.coli and yeast cell culture were approved for clinical applications. For their production in the large amount and cheaper expense, the method using the milk of transgenic animal, so called animal bioreactor, was introduced. For mammary gland-specific expression of human GM-CSF in the transgenic animal, goat β-casein promoter was used in the construction of expression cassette. Two fragment in the three in various length, 0.72kb, 1.76kb were amplified by polymerase chain reaction from goat genomic DNA and one, 6.18kb, was prepared from pBC1 vector by restriction-enzyme digestion. And human GM-CSF structural gene was also amplified by polymerase chain reaction from human genomic DNA. First of all, to make β0.72pGbc-hGMCSF, I subcloned human GM-CSF structural gene in front of bovine growth hormone (BGH) terminator of pRc/RSV vector and then subcloned 0.72kb goat β-casein promoter fragment before human GM-CSF structural gene. And for β1.76pGbc-hGMCSF 1.76kb goat β-casein promoter fragment was subcloned in pBluescriptII SK and human GM-CSF structural gene and BGH terminator part which was prepared from β0.72pGbc-hGMCSF by restriction enzyme was subcloned behind 1.76 goat β-casein promoter fragment. Also for β6.18pGbc-hGMCSF human GM-CSF structural gene and BGH terminator part which...