A 3,387 bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice
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A new expression vector containing the 1,944 bp 5′-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3′-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19–40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.
KeywordsPromoter of alpha-S1-casein gene Tissue-specific expression Mammary gland Human granulocyte colony-stimulating factor Transgenic mice
The authors express gratitude to Andrey Slobodskoy, Helen Lassunskaya (State Univeristy of North Fluminense, Campos, RJ, Brazil) for Western blotting, Nelly Khaidarova (Institute of Molecular Genetics, Academy of Sciences of Russia, Moscow, Russia), Hélio Menezes (Institute of Anatomy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil) for help in preparation recombinant DNA and determination of biological activity of hG-CSF.
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