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Nucleic Acids Research
Barash, I., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Nathan, M., Rhone Poulenc Rorer Central Research, 500 Arcola Road, Collegeville, PA 19426, United States, Life Technologies, PO Box 6009, Gaithesburg, MD 20877, United States
Kari, R., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Ilan, N., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Shani, M., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Hurwitz, D.R., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel, ALG Company, 734 Forest Street, Marlboro, MA 01752, United States
Two new β-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5′- or 3′-intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG-directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3′ polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1,195-208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I., Faerman,A., Ratovitsky,T., Puzis,R., Nathan,M. Hurwitz,D.R. and Shani,M. (1994) Transgenic Res. 3,141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.
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הספר "אוצר וולקני"
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תנאי שימוש
Elements within the β-lactoglobulin gene inhibit expression of human serum albumin cDNA minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice
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Barash, I., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Nathan, M., Rhone Poulenc Rorer Central Research, 500 Arcola Road, Collegeville, PA 19426, United States, Life Technologies, PO Box 6009, Gaithesburg, MD 20877, United States
Kari, R., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Ilan, N., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Shani, M., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel
Hurwitz, D.R., Institute of Animal Science, Volcani Center, Bet Dagan 50250, Israel, ALG Company, 734 Forest Street, Marlboro, MA 01752, United States
Elements within the β-lactoglobulin gene inhibit expression of human serum albumin cDNA minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice
Two new β-lactoglobulin (BLG)/human serum albumin (HSA) hybrid gene vectors were constructed and tested for expression in COS-7 cells and in transgenic mice. The HSA sequences were inserted between the second and sixth BLG exons. Transient transfection experiments with these vectors as well as a series of additional vectors with either the BLG 5′- or 3′-intragenic sequences revealed that sequences within BLG exon 1/intron 1/exon 2 abrogated BLG-directed HSA expression in vitro, regardless of the presence of HSA introns or the origin of the 3′ polyadenylation signal. In contrast, the same BLG expression cassette enabled the efficient expression of HSA cDNA or minigene in the mammary gland of transgenic mice with subsequent secretion of the corresponding protein into the milk of 56 and 82%, respectively of the mouse strains at levels up to 0.3 mg/ml. Previous attempts to express HSA cDNA inserted into exon 1 of the BLG gene had failed [Shani,M., Barash,I., Nathan,M., Ricca,G., Searfoss,G.H., Dekel,I., Faerman,A., Givol,D. and Hurwitz,D.R. (1992) Transgenic Res. 1,195-208]. The new BLG expression cassette conferred more stringent tissue specific expression than previously described BLG/HSA constructs [Barash,I., Faerman,A., Ratovitsky,T., Puzis,R., Nathan,M. Hurwitz,D.R. and Shani,M. (1994) Transgenic Res. 3,141-151]. However, it was not able to insulate the transgenes from the surrounding host DNA sequences and did not result in copy number dependent expression in transgenics. Together, the in vitro and in vivo results suggest both positive and negative regulatory elements within the BLG intragenic sequences evaluated. The new BLG construct represents an extremely valuable vector for the efficient expression of cDNAs in the mammary gland of transgenic animals.
Scientific Publication
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