Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice

In developing mouse embryos, MyoD family regulatory genes are expressed specifically in muscle precursors and mature myofibers. This pattern, taken together with the well-established ability of MyoD family members to convert a variety of cell types to skeletal muscle, suggests a significant role for these genes in regulating skeletal myogenesis. The possibility that expression of these genes may be causally associated with segregation of the myogenic lineage from other mesodermal derivatives, or with the subsequent maintenance of muscle phenotypes at later times, raises the issue of how MyoD family genes are themselves regulated during development. In this work, we have initiated studies to identify DNA sequences that govern Myf-5 and MRF4 (herculin, myf-6) transcription. Myf-5 is the first of the MyoD family to be expressed in the developing mouse embryo, while MRF4 is the most abundantly expressed myogenic factor in postnatal animals. In spite of their strikingly divergent patterns of expression, Myf-5 and MRF4 are tightly linked in the mouse genome; their translational start codons are only 8.5 kilobases apart. Here, the 5′ flanking regions of the mouse Myf-5 and MRF4 genes were separately linked to a bacterial beta-galactosidase (lacZ) gene, and these constructs were each used to produce several lines of transgenic mice. Transgene expression was monitored by X-gal staining of whole embryos and by in situ hybridization of embryo sections. For the Myf-5/lacZ lines, the most intense transgene expression was in the visceral arches and their craniofacial muscle derivatives, beginning at day 8.75 post coitum (p.c.). This correlates with endogenous Myf-5 expression in visceral arches.(ABSTRACT TRUNCATED AT 250 WORDS)

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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 ◽

10.1182/blood.v85.2.319.319 ◽

1995 ◽

Vol 85 (2) ◽

pp. 319-329 ◽

Cited By ~ 66

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3978-3990.1992 ◽

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.

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Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.1.f54 ◽

2001 ◽

Vol 280 (1) ◽

pp. F54-F60 ◽

Cited By ~ 38

Author(s):

Yueming Ding ◽

Curt D. Sigmund

Keyword(s):

Transgenic Mice ◽

Hormonal Regulation ◽

Transgene Expression ◽

Proximal Tubule Cell ◽

Dependent Manner ◽

Specific Expression ◽

Androgen Receptor Antagonist ◽

Sexually Dimorphic Expression ◽

Androgen Regulation ◽

High Doses

We previously reported a novel transgenic model expressing human angiotensinogen from the kidney androgen-regulated protein promoter, and demonstrated sexually dimorphic expression. Herein, we investigated the hormonal regulation of this transgene. Testosterone increased transgene expression in female mice in a dose- and time-dependent manner and was not detectable 3-days after treatment was halted. High doses of estrogen were required to induce the transgene. Expression of transgene mRNA decreased after castration of male transgenic mice. As in females, however, transgene expression could be induced after administration of testosterone. Flutamide, an androgen receptor antagonist, dose dependently blocked transgene expression in males and blunted the induction caused by testosterone in females. Neither testosterone nor estrogen altered the proximal tubule cell-specific expression of the transgene. The data suggest that the level of transgene expression in this model can be controlled temporally and in magnitude by manipulating the levels of androgen. The fortuitous androgen regulation of this transgene can be used as a molecular “on-off” switch to control transgene expression and potentially manipulate blood pressure levels in this model.

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Rescue of Prothrombin-deficiency by Transgene Expression in Mice

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613344 ◽

2002 ◽

Vol 88 (12) ◽

pp. 984-991 ◽

Cited By ~ 20

Author(s):

William Sun ◽

Mallory Coleman ◽

David Witte ◽

Sandra Degen

Keyword(s):

Transgenic Mice ◽

Transgene Expression ◽

Embryonic Lethality ◽

The Other ◽

Model Systems ◽

Bleeding Events ◽

Specific Expression ◽

Spontaneous Bleeding ◽

Knock Out ◽

Transgenic Expression

SummaryProthrombin has diverse biological functions in addition to its well established role in blood coagulation. In order to study these functions in more detail mouse model systems are needed. Since deficiency of prothrombin in mice results in partial embryonic lethality and neonatal death, alternative approaches are required to study the biology of prothrombin in the adult mouse. The liver is the major site of synthesis of prothrombin and therefore liver-specific promoters were used to express prothrombin in transgenic mice. Mice generated from crosses with these transgenic mice and mice hemizygous for the knock-out allele were used to test whether liver-specific expression is sufficient to correct the phenotype of null mice and whether liver-specific expression is sufficient for the development and survival of mice to adulthood. The mouse albumin promoter/enhancer was used initially for transgene expression without success in obtaining transgene positive, endogenous prothrombin null mice. Two lines of transgene positive, endogenous prothrombin deficient mice were obtained using the mouse transthyretin (TTR) promoter/enhancer driving expression of a human prothrombin cDNA. One line was able to rescue both the embryonic and the neonatal lethality while the other line was only able to correct the embryonic lethality. Expression of prothrombin was restricted to the liver and stomach in one line and to the liver, pancreas, stomach and kidney in the other line of mice. Thrombin activity for one line was determined to be at 5-10% of wildtype levels. These mice developed normally and did not have spontaneous bleeding events unless traumatized. Therefore, transgenic expression of human prothrombin is sufficient for the rescue of the lethality found for prothrombin deficiency in mice.

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Cell-Type-Specific Expression of a TESK1 Promoter-Linked lacZ Gene in Transgenic Mice

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2001.5404 ◽

2001 ◽

Vol 286 (3) ◽

pp. 566-573 ◽

Cited By ~ 18

Author(s):

Jiro Toshima ◽

Junko Y. Toshima ◽

Misao Suzuki ◽

Tetsuo Noda ◽

Kensaku Mizuno

Keyword(s):

Transgenic Mice ◽

Lacz Gene ◽

Cell Type ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Cell Type Specific

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Promoter Elements of vav Drive Transgene Expression In Vivo Throughout the Hematopoietic Compartment

Blood ◽

10.1182/blood.v94.6.1855.418k33_1855_1863 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1855-1863 ◽

Cited By ~ 7

Author(s):

Sarah Ogilvy ◽

Donald Metcalf ◽

Leonie Gibson ◽

Mary L. Bath ◽

Alan W. Harris ◽

...

Keyword(s):

Reporter Gene ◽

Transgene Expression ◽

Cell Types ◽

Transcription Unit ◽

Erythroid Cells ◽

Promoter Elements ◽

Specific Expression ◽

Expression Of Genes ◽

B And T Lymphocytes

To develop a method for targeting expression of genes to the full hematopoietic system, we have used transgenic mice to explore the transcriptional regulation of the vav gene, which is expressed throughout this compartment but rarely outside it. Previously, we showed that a cluster of elements surrounding its promoter could drive hematopoietic-specific expression of a bacterial lacZ reporter gene, but the expression was confined to lymphocytes and was sporadically silenced. Those limitations are ascribed here to the prokaryotic reporter gene. With a human CD4 (hCD4) cell surface reporter, the vav promoter elements drove expression efficiently and stably in virtually all nucleated cells of adult hematopoietic tissues but not notably in nonhematopoietic cell types. In multiple lines, hCD4 appeared on most, if not all, B and T lymphocytes, granulocytes, monocytes, megakaryocytes, eosinophils, and nucleated erythroid cells. Moreover, high levels appeared on both lineage-committed progenitors and the more primitive preprogenitors. In the fetus, expression was evident in erythroid cells of the definitive but not the primitive type. These results indicate that a prokaryotic sequence can inactivate a transcription unit and that the vavpromoter region constitutes a potent transgenic vector for the entire definitive hematopoietic compartment.

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The Vascular Endothelial-Cadherin Promoter Directs Endothelial-Specific Expression in Transgenic Mice

Blood ◽

10.1182/blood.v93.1.184.401a20_184_192 ◽

1999 ◽

Vol 93 (1) ◽

pp. 184-192 ◽

Cited By ~ 4

Author(s):

S. Gory ◽

M. Vernet ◽

M. Laurent ◽

E. Dejana ◽

J. Dalmon ◽

...

Keyword(s):

Endothelial Cells ◽

Transgenic Mice ◽

Dna Sequences ◽

Vascular System ◽

Proximal Region ◽

Vascular Endothelial ◽

Specific Expression ◽

Functional Regions ◽

Vascular Endothelial Cadherin

Vascular endothelial-cadherin (VE-cadherin) is a calcium-dependent adhesive molecule, exclusively and constitutively expressed in endothelial cells. Analysis of the VE-cadherin promoter fused to a reporter gene in bovine aortic endothelial cells showed three major functional regions. The proximal region alone (−139, +24) promoted nonspecific transcription; the addition of the (−289, −140) and (−2226, −1190) domains abolished transcription in fibroblasts while expression in endothelial cells remained unchanged, suggesting that fragments (−2226, +24) and longer contain the full endogenous promoter activity. To study the transcriptional specificity of the promoter region in vivo, we generated transgenic mice carrying the chimeric construct containing the (−2486, +24) region. The promoter directed reporter expression in all examined organs of adult transgenic mice. During embryonic development, transgene expression was detected at the early steps of vasculogenesis. Later, the expression persisted during development of the vascular system and was restricted to the endothelial layer of the vessels. Together, these data provide evidence for specific regulatory regions within the VE-cadherinpromoter. Furthermore, the identification of DNA sequences restricting gene expression to the endothelium has many potential applications for the development of animal models of cardiovascular or angiogenic diseases or for the delivery of therapeutic molecules.

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A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽

10.1182/blood.v96.9.3040.h8003040_3040_3048 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3040-3048

Author(s):

Ahlke Heydemann ◽

Soren Warming ◽

Cynthia Clendenin ◽

Kirsten Sigrist ◽

J. Peter Hjorth ◽

...

Keyword(s):

Transgenic Mice ◽

Dna Sequences ◽

Copy Number ◽

Messenger Rna ◽

Transgene Expression ◽

Fluorescent Protein ◽

Control Function ◽

Integration Site ◽

Myeloid Cells ◽

Independent Copy

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

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A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽

10.1182/blood.v96.9.3040 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3040-3048 ◽

Cited By ~ 15

Author(s):

Ahlke Heydemann ◽

Soren Warming ◽

Cynthia Clendenin ◽

Kirsten Sigrist ◽

J. Peter Hjorth ◽

...

Keyword(s):

Transgenic Mice ◽

Dna Sequences ◽

Copy Number ◽

Messenger Rna ◽

Transgene Expression ◽

Fluorescent Protein ◽

Control Function ◽

Integration Site ◽

Myeloid Cells ◽

Independent Copy

Abstract The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

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