Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region
Identifieur interne : 000316 ( France/Analysis ); précédent : 000315; suivant : 000317Human insulin gene expression in transgenic mice: mutational analysis of the regulatory region
Auteurs : Jean-Michel Itier [France] ; Philippe Douhet [France] ; Pierrette Desbois [France] ; Rajiv L. Joshi [France] ; Franoise Dandoy-Dron [France] ; Jacques Jami [France] ; Danielle Bucchini [France]Source :
- Differentiation [ 0301-4681 ] ; 1996.
English descriptors
- Teeft :
- Acad, Cell biol, Cell specificity, Ctii, Ctii motifs, Ctiii, Deletion, Edlund, Efficient expression, Enhancer, Enhancer activity, Exon, Gcii, Gene, Gene expression, Human insulin gene, Human insulin gene expression, Human insulin transcripts, Insulin, Insulin gene, Insulinoma, Jami, Monoclonal antibody, Mouse, Mutation, Mutational analysis, Mutii, Mutiii, Mutiv, Natl, Northern blot analysis, Nucleic acids, Pancreas, Pancreatic, Pancreatic sections, Present work, Proc, Proc natl acad, Promoter, Promoter region, Reporter gene, Room temperature, Rutter, Transcription, Transcriptional, Transcriptional regulation, Transfected, Transfected insulinoma cells, Transgene, Transgene expression, Transgenic, Transgenic controls, Transgenic lines, Transgenic mice, Transgenic mouse lines, Urine samples.
Abstract
Abstract: A mini-human insulin gene and four derivatives mutated at several regions potentially involved in the regulation of gene expression were used to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied using three approaches: (1) Northern blot analysis using total RNA from pancreas and other organs, (2) radioimmunoassay to detect the human C-peptide in urine samples, and (3) immunocytochemistry of pancreas sections to examine whether expression of the transgene was still specifically expressed in -cells. Mutation of the cis-acting elements located between 238 and 206 (GCII and CTII motifs) resulted in a strong decrease of gene expression in the pancreas of transgenic mice, but it did not lead to complete extinction of the transgene expression. This region alone (255202), when linked to the minimal Herpes simplex virus thymidine kinase gene (tk) promoter, failed to activate chloramphenicol acetyltransferase (CAT) gene expression in transfected insulinoma cells, while it was activated by the equivalent region of the rat insulin I gene. On the contrary, mutation of the DNA motifs located between 109 and 75 (GCI and CTI) or between 323 and 297 (CTIII) did not significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (581) by the tk promoter did not alter its level of expression in transgenic mice. In all instances, expression of the different transgenes remained localized in the islet -cells. Altogether, these results indicate that the GCII-CTII motif is an important regulatory element for efficient expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of other elements.
Url:
DOI: 10.1046/j.1432-0436.1996.6050309.x
Affiliations:
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<term>Ctiii</term>
<term>Deletion</term>
<term>Edlund</term>
<term>Efficient expression</term>
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<term>Enhancer activity</term>
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<term>Gene expression</term>
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<term>Human insulin gene expression</term>
<term>Human insulin transcripts</term>
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<term>Insulin gene</term>
<term>Insulinoma</term>
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<term>Monoclonal antibody</term>
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<term>Mutation</term>
<term>Mutational analysis</term>
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<term>Mutiv</term>
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<term>Northern blot analysis</term>
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<term>Transcription</term>
<term>Transcriptional</term>
<term>Transcriptional regulation</term>
<term>Transfected</term>
<term>Transfected insulinoma cells</term>
<term>Transgene</term>
<term>Transgene expression</term>
<term>Transgenic</term>
<term>Transgenic controls</term>
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<front><div type="abstract" xml:lang="en">Abstract: A mini-human insulin gene and four derivatives mutated at several regions potentially involved in the regulation of gene expression were used to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied using three approaches: (1) Northern blot analysis using total RNA from pancreas and other organs, (2) radioimmunoassay to detect the human C-peptide in urine samples, and (3) immunocytochemistry of pancreas sections to examine whether expression of the transgene was still specifically expressed in -cells. Mutation of the cis-acting elements located between 238 and 206 (GCII and CTII motifs) resulted in a strong decrease of gene expression in the pancreas of transgenic mice, but it did not lead to complete extinction of the transgene expression. This region alone (255202), when linked to the minimal Herpes simplex virus thymidine kinase gene (tk) promoter, failed to activate chloramphenicol acetyltransferase (CAT) gene expression in transfected insulinoma cells, while it was activated by the equivalent region of the rat insulin I gene. On the contrary, mutation of the DNA motifs located between 109 and 75 (GCI and CTI) or between 323 and 297 (CTIII) did not significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (581) by the tk promoter did not alter its level of expression in transgenic mice. In all instances, expression of the different transgenes remained localized in the islet -cells. Altogether, these results indicate that the GCII-CTII motif is an important regulatory element for efficient expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of other elements.</div>
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