Production of human colony stimulating factors using goat beta-casein promoter

Abstract

Human granulocyte colony stimulating factor (G-CSF) and human granulocyte macrophage colony stimulating factor (GM-CSF) are the most widely used human hemopoietic factors to remedy different forms of neutropenia, chemotherapy-induced leucopenia, and in the mobilization of progenitor cells for autologous or allogenic transplantation. These colony stimulating factors maintain 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 CHO cell culture were approved for clinical application. For their production in large amount and cheaper expense, the method using milk of transgenic animal, so called animal bioreactor, was introduced. For mammary gland-specific expression of human CSFs in transgenic animals, goat β-casein promoter was used in the construction of expression cassette. Four fragments in various length, 0.6 kb, 1.7 kb, 5.5 kb, and 7.0 kb, were amplified by polymerase chain reaction from a 9.3 kb fragment containing the known goat β-casein promoter and truncated exon 1 and subcloned into pBluescript II SK(+) plasmid. Human G-CSF and GM-CSF gene were subcloned in front of bovine growth hormone (BGH) terminator of pRc/RSV vector, and then the fragments containing CSF genes and BGH terminator were subcloned behind the goat β-casein promoters. These constructs were named as pGbc0.6-hGCSF/GMCSF, pGbc-hGCSF/GMCSF, pGbc5.5-hGCSF, and pGbc7.0-hGCSF according to the length of goat β-casein promoter and the name of the structural gene. The constructed vectors were introduced into HC11 cell, a mammary gland originated epithelial cell line, by stable transfection to verify expression of human CSFs under direction by goat β-casein promoter. ...