Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice

1995 ◽  
Vol 108 (2) ◽  
pp. 811-820 ◽  
Author(s):  
L. Casanova ◽  
A. Bravo ◽  
F. Were ◽  
A. Ramirez ◽  
J.J. Jorcano ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
The Body ◽  
Gene Copy ◽  
Intermediate Filament Protein ◽  
Lacz Gene ◽  
Tissue Specific ◽  
Endogenous Gene ◽  
Flanking Sequences ◽  
Dna Fragment ◽  
Keratin 8

Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5′ flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3′ flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5′ and 3′ flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene.

Transcriptional insulation of the human keratin 18 gene in transgenic mice

1993 ◽  
Vol 13 (4) ◽  
pp. 2214-2223
Author(s):  
N Neznanov ◽  
I S Thorey ◽  
G Ceceña ◽  
R G Oshima
Keyword(s):  
Transgenic Mice ◽  
Thymidine Kinase ◽  
Copy Number ◽  
Thymidine Kinase Gene ◽  
Flanking Sequence ◽  
Kinase Gene ◽  
Tissue Specific ◽  
Dependent Behavior ◽  
Flanking Sequences ◽  
Keratin 18

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes.

scholarly journals Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells.

1989 ◽  
Vol 108 (3) ◽  
pp. 1009-1024 ◽  
Author(s):  
F R Pieper ◽  
G Schaart ◽  
P J Krimpenfort ◽  
J B Henderik ◽  
H J Moshage ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Intermediate Filament ◽  
Germ Line ◽  
Fibroblast Cell ◽  
Intermediate Filament Protein ◽  
Coding Region ◽  
Tissue Specific ◽  
Nonmuscle Cells ◽  
Filament Protein ◽  
Vimentin Gene

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.

Tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein in transgenic mice

1989 ◽  
Vol 9 (5) ◽  
pp. 2254-2257
Author(s):  
J Allison ◽  
Y L Zhang ◽  
M G Parker
Keyword(s):  
Transgenic Mice ◽  
Hormonal Regulation ◽  
Binding Protein ◽  
Ventral Prostate ◽  
Tissue Specific ◽  
Regulated Expression ◽  
Selective Expression ◽  
Flanking Sequences ◽  
Steroid Binding ◽  
Steroid Binding Protein

We investigated the tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein by introducing the C3(1) gene with 4-kilobase (kb) upstream and 2-kb downstream flanking sequences into transgenic mice. There was selective expression in the ventral prostate that was stimulated by testosterone, which indicated that the gene together with 6-kb flanking DNA contains the information required for prostate-specific and testosterone-regulated expression.

scholarly journals Transgenic mice carrying the erythropoietin gene promoter linked to lacZ express the reporter in proximal convoluted tubule cells after hypoxia

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1831-1836 ◽  
Author(s):  
F Loya ◽  
Y Yang ◽  
H Lin ◽  
E Goldwasser ◽  
M Albitar
Keyword(s):  
Transgenic Mice ◽  
Gene Promoter ◽  
Large Degree ◽  
Proximal Convoluted Tubule ◽  
Lacz Gene ◽  
Lacz Expression ◽  
Specific Expression ◽  
Tubular Cells ◽  
Dna Fragment ◽  
Significant Expression

Results on the localization of erythropoietin (Epo) synthesis in renal cells have been contradictory, implicating either interstitial or tubular cells. We fused the lacZ gene to a 7-kb DNA fragment of the mouse Epo gene encompassing a portion of the first intron, the first exon, and a 6-kb sequence of the 5′-flanking region. Transgenic mice carrying this construct show a low level of specific expression of the lacZ gene in proximal convoluted tubule (PCT) cells. Without hypoxia, no significant expression was detected in the liver. Hypoxia induced a large degree of lacZ expression, mainly in kidney PCT cells and to a lesser degree in liver. However, anemia induced lacZ expression in both kidney and liver. These findings indicate that, under these conditions, Epo is expressed in tubular cells, specifically PCT cells.

scholarly journals Transgenic mice carrying the erythropoietin gene promoter linked to lacZ express the reporter in proximal convoluted tubule cells after hypoxia

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1831-1836 ◽  
Author(s):  
F Loya ◽  
Y Yang ◽  
H Lin ◽  
E Goldwasser ◽  
M Albitar
Keyword(s):  
Transgenic Mice ◽  
Gene Promoter ◽  
Large Degree ◽  
Proximal Convoluted Tubule ◽  
Lacz Gene ◽  
Lacz Expression ◽  
Specific Expression ◽  
Tubular Cells ◽  
Dna Fragment ◽  
Significant Expression

Abstract Results on the localization of erythropoietin (Epo) synthesis in renal cells have been contradictory, implicating either interstitial or tubular cells. We fused the lacZ gene to a 7-kb DNA fragment of the mouse Epo gene encompassing a portion of the first intron, the first exon, and a 6-kb sequence of the 5′-flanking region. Transgenic mice carrying this construct show a low level of specific expression of the lacZ gene in proximal convoluted tubule (PCT) cells. Without hypoxia, no significant expression was detected in the liver. Hypoxia induced a large degree of lacZ expression, mainly in kidney PCT cells and to a lesser degree in liver. However, anemia induced lacZ expression in both kidney and liver. These findings indicate that, under these conditions, Epo is expressed in tubular cells, specifically PCT cells.

scholarly journals An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

1991 ◽  
Vol 11 (4) ◽  
pp. 2066-2074 ◽  
Author(s):  
J L Slack ◽  
D J Liska ◽  
P Bornstein
Keyword(s):  
Transgenic Mice ◽  
Regulatory Region ◽  
Collagen Gene ◽  
Flanking Sequence ◽  
Specific Expression ◽  
Tissue Specific ◽  
Endogenous Gene ◽  
Tissue Specific Expression ◽  
First Intron ◽  
High Level

Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein in transgenic mice.

1989 ◽  
Vol 9 (5) ◽  
pp. 2254-2257 ◽  
Author(s):  
J Allison ◽  
Y L Zhang ◽  
M G Parker
Keyword(s):  
Transgenic Mice ◽  
Hormonal Regulation ◽  
Binding Protein ◽  
Ventral Prostate ◽  
Tissue Specific ◽  
Regulated Expression ◽  
Selective Expression ◽  
Flanking Sequences ◽  
Steroid Binding ◽  
Steroid Binding Protein

We investigated the tissue-specific and hormonal regulation of the gene for rat prostatic steroid-binding protein by introducing the C3(1) gene with 4-kilobase (kb) upstream and 2-kb downstream flanking sequences into transgenic mice. There was selective expression in the ventral prostate that was stimulated by testosterone, which indicated that the gene together with 6-kb flanking DNA contains the information required for prostate-specific and testosterone-regulated expression.

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