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פותח על ידי קלירמאש פתרונות בע"מ -
A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice
Year:
2001
Source of publication :
Transgenic Research
Authors :
ברש, איתמר
;
.
רייכנשטיין, משה
;
.
Volume :
10
Co-Authors:
Reichenstein, M., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Gottlieb, H., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Damari, G.-M., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Iavnilovitch, E., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Barash, I., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Facilitators :
From page:
445
To page:
456
(
Total pages:
12
)
Abstract:
A β-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3′ region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3′ end, with BLG sequences of exon 6/intron 6/exon 7 and the 3′ region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3′ flanking sequences. The BLG 5′ regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.
Note:
Related Files :
Animals
Female
gene expression
Mammary gland
mice
Rodentia
Serum Albumin
עוד תגיות
תוכן קשור
More details
DOI :
10.1023/A:1012064922126
Article number:
Affiliations:
Database:
סקופוס
Publication Type:
מאמר
;
.
Language:
אנגלית
Editors' remarks:
ID:
32744
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 01:12
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Scientific Publication
A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice
10
Reichenstein, M., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Gottlieb, H., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Damari, G.-M., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Iavnilovitch, E., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
Barash, I., Institute of Animal Science, Agricultural Research Organization, Volcani Center, Bet Dagan, Israel
A new β-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice
A β-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3′ region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3′ end, with BLG sequences of exon 6/intron 6/exon 7 and the 3′ region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3′ flanking sequences. The BLG 5′ regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.
Scientific Publication
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