Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 947-959 ◽  
Author(s):  
Z. Li ◽  
P. Marchand ◽  
J. Humbert ◽  
C. Babinet ◽  
D. Paulin
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Skeletal Muscles ◽  
Regulatory Elements ◽  
Limb Bud ◽  
Regulatory Sequence ◽  
Cardiac Muscle Cells ◽  
Specific Expression ◽  
Foetal Development ◽  
Beta Galactosidase

During the development of the mouse embryo, desmin is one of the first muscle proteins detected in both the heart and the somites. The expression of the desmin gene differs from most other muscle genes, since it is initiated in replicating myoblasts and accumulates as the muscle differentiates. We have characterized a muscle-specific enhancer which directs the expression of desmin in vitro in the myoblasts and myotubes of C2 cells but not in non-myogenic cells. We report here on the generation and characterization of transgenic mice bearing a transgene in which the 1 kb DNA 5′ regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coli beta-galactosidase (Des1-nlacZ). The enhancer activity of the desmin promoter is very strong and the reporter gene expression is easily detected in tissue sections. We have demonstrated that the regulatory elements present in the transgene Des1-nlacZ are sufficient to direct muscle-specific and developmentally regulated expression of nlacZ in skeletal muscles. Endogenous desmin expression and transgene activity were found to be correlated during the development of skeletal muscles. The transgene was expressed in the committed mononucleate myoblasts as well as in the myotubes. In addition, we have shown that the desmin-derived sequences direct a highly selective expression of nlacZ in cells that leave the somites and invade the limb bud, indicating that the cells that migrate from the somites are already predetermined for myogenesis. In contrast, smooth and cardiac muscle cells were beta-galactosidase negative both during embryonic and foetal development. Interestingly, the transgene was found to be expressed in the conduction system of the heart, which exhibits many features characteristic of skeletal muscles.

A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice

1995 ◽  
Vol 108 (12) ◽  
pp. 3677-3684 ◽  
Author(s):  
G. Zhou ◽  
S. Garofalo ◽  
K. Mukhopadhyay ◽  
V. Lefebvre ◽  
C.N. Smith ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Type Ii Collagen ◽  
Mouse Embryos ◽  
Collagen Gene ◽  
Specific Expression ◽  
Intact Mouse ◽  
Endogenous Gene ◽  
Intron 1 ◽  
Beta Galactosidase

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Fast skeletal muscle-specific expression of a quail troponin I gene in transgenic mice.

1988 ◽  
Vol 8 (12) ◽  
pp. 5072-5079 ◽  
Author(s):  
P L Hallauer ◽  
K E Hastings ◽  
A C Peterson
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Troponin I ◽  
Fiber Type ◽  
Regulatory Elements ◽  
Evolutionary Divergence ◽  
Mrna Levels ◽  
Fast Skeletal Muscle ◽  
Specific Expression ◽  
Exon 2

We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.

scholarly journals A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1072-1074 ◽  
Author(s):  
Sandra Ziegler ◽  
Kurt Bürki ◽  
Radek C. Skoda
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Fetal Liver ◽  
Regulatory Elements ◽  
Yolk Sac ◽  
Placental Alkaline Phosphatase ◽  
Hematopoietic Progenitors ◽  
Thrombopoietin Receptor ◽  
Human Placental Alkaline Phosphatase ◽  
Dna Fragment

Abstract Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5′-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis.

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

scholarly journals Specific expression of the human CD4 gene in mature CD4+ CD8- and immature CD4+ CD8+ T cells and in macrophages of transgenic mice

1994 ◽  
Vol 14 (2) ◽  
pp. 1084-1094
Author(s):  
Z Hanna ◽  
C Simard ◽  
A Laperrière ◽  
P Jolicoeur
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Transcription Initiation ◽  
Critical Role ◽  
Regulatory Elements ◽  
T Cell Subsets ◽  
Double Positive ◽  
Specific Expression

The CD4 protein plays a critical role in the development and function of the immune system. To gain more insight into the mechanism of expression of the human CD4 gene, we cloned 42.2 kbp of genomic sequences comprising the CD4 gene and its surrounding sequences. Studies with transgenic mice revealed that a 12.6-kbp fragment of the human CD4 gene (comprising 2.6 kbp of 5' sequences upstream of the transcription initiation site, the first two exons and introns, and part of exon 3) contains the sequences required to support the appropriate expression in murine mature CD4+ CD8- T cells and macrophages but not in immature double-positive CD4+ CD8+ T cells. Expression in CD4+ CD8+ T cells was found to require additional regulatory elements present in a T-cell enhancer fragment recently identified for the murine CD4 gene (S. Sawada and D. R. Littman, Mol. Cell. Biol. 11:5506-5515, 1991). These results suggest that expression of CD4 in mature and immature T-cell subsets may be controlled by distinct and independent regulatory elements. Alternatively, specific regulatory elements may control the expression of CD4 at different levels in mature and immature T-cell subsets. Our data also indicate that mouse macrophages contain the regulatory factors necessary to transcribe the human CD4 gene.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

1997 ◽  
Vol 17 (10) ◽  
pp. 6002-6013 ◽  
Author(s):  
K L Wu ◽  
M Gannon ◽  
M Peshavaria ◽  
M F Offield ◽  
E Henderson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Beta Cell ◽  
Beta Cells ◽  
Cell Lines ◽  
Pancreatic Beta Cell ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Specific Expression ◽  
Islet Beta Cells ◽  
Beta Galactosidase

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.

Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines

1995 ◽  
Vol 269 (6) ◽  
pp. G925-G939 ◽  
Author(s):  
A. J. Markowitz ◽  
G. D. Wu ◽  
A. Bader ◽  
Z. Cui ◽  
L. Chen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Lines ◽  
Reporter Gene ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Transgenic Animals ◽  
Intestinal Cell ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

scholarly journals Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

1990 ◽  
Vol 10 (11) ◽  
pp. 5822-5829 ◽  
Author(s):  
S R Ross ◽  
C L Hsu ◽  
Y Choi ◽  
E Mok ◽  
J P Dudley
Keyword(s):  
Transgenic Mice ◽  
Mouse Mammary Tumor Virus ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Mmtv Ltr ◽  
Inappropriate Expression

Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene.

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