scholarly journals 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.

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 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 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

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.

Reporter gene expression in cones in transgenic mice carrying bovine rhodopsin promoter/lacZ transgenes

1994 ◽  
Vol 11 (6) ◽  
pp. 1227-1231 ◽  
Author(s):  
Peter Gouras ◽  
Hild Kjeldbye ◽  
Donald J. Zack
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Messenger Rna ◽  
Reporter Gene Expression ◽  
Specific Expression ◽  
Cis Acting ◽  
Bovine Rhodopsin ◽  
Transgenic Lines ◽  
Dna Elements

AbstractRhodopsin gene expression has been used as a model system to study the mechanisms regulating photoreceptor gene expression. Previous transgenic experiments using rhodopsin promoter/lacZ fusion constructs identified some of the cis-acting DNA elements responsible for photoreceptor cell-specific expression. However, the issue of rod specificity vs. photoreceptor (rod and cone) specificity of the elements was not resolved. To address this issue, the specificity of reporter gene expression in the retinas of transgenic mice carrying bovine rhodopsin promoter/lacZ (ß-galactosidase) fusion genes was assessed using X-gal staining and electron microscopy. Two independent transgenic lines, one carrying a rhodopsin promoter fragment extending from −2174 to +70 base pairs (bp) relative to the messenger RNA start site and another line carrying a fragment from −222 to +70 bp, both showed reporter gene expression in cones as well as rods, although the level of staining appeared to be less in the cones than in the rods. These results demonstrate that the −2174 to +70 bp and −222 to +70 bp bovine rhodopsin promoter fragments are not rod-specific in transgenic mice and indicate that the existence of rod promoter mediated-expression in cones must be considered when interpreting results from transgenic experiments utilizing the rhodopsin promoter.

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.

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

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 1855-1863 ◽  
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.

scholarly journals Leukocyte integrin CD11b promoter directs expression in lymphocytes and granulocytes in transgenic mice

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 1017-1024 ◽  
Author(s):  
A Back ◽  
K East ◽  
D Hickstein
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Human Leukocyte ◽  
Surface Expression ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Integrin Subunit ◽  
Cd11b Expression

The human leukocyte integrin subunit CD11b is expressed predominantly on myelomonocytic cells. To identify CD11b promoter sequences important for myelomonocytic gene expression and to assess the utility of the CD11b promoter for expressing heterologous genes in vivo, we generated transgenic mice with a human CD4 reporter gene driven by CD11b promoter constructs composed of 1.5, 0.3, or 0.1 kb of DNA sequence 5′ to the transcription start site. Using flow cytometry to detect the human CD4 reporter on murine leukocytes, two of three 1.5-kb CD11b promoter founder lines showed surface expression of the human CD4 transgene in granulocytes and lymphocytes. The transgene expression observed in lymphocytes was inappropriate relative to the normal pattern of CD11b expression. Of the eight 0.3-kb or 0.1-kb founder lines, only one 0.1- kb founder line showed transgene expression. The overall pattern of transgene expression among the 11 founder lines does not parallel expression of the endogenous CD11b gene. These studies indicate that additional CD11b regulatory elements will be required to express a reporter gene in vivo in a lineage-specific pattern that mimics the endogenous CD11b gene.

scholarly journals Potential Autoregulation of Transcription Factor PU.1 by an Upstream Regulatory Element

2005 ◽  
Vol 25 (7) ◽  
pp. 2832-2845 ◽  
Author(s):  
Yutaka Okuno ◽  
Gang Huang ◽  
Frank Rosenbauer ◽  
Erica K. Evans ◽  
Hanna S. Radomska ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Reporter Gene ◽  
Regulatory Element ◽  
Mammalian Species ◽  
Critical Role ◽  
Homologous Region ◽  
Specific Expression ◽  
Upstream Regulatory Element

ABSTRACT Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb −14 site confers lineage-specific reporter gene expression in vivo. This kb −14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

scholarly journals DNA elements with AT-rich core sequences direct pituitary cell-specific expression of the pro-opiomelanocortin gene in transgenic mice

1995 ◽  
Vol 312 (3) ◽  
pp. 827-832 ◽  
Author(s):  
B Liu ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Protein Interactions ◽  
Transcriptional Activator ◽  
Pituitary Tumour ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Specific Gene ◽  
Specific Expression ◽  
Pomc Gene

Corticotrophs are the first fully differentiated cells to appear in the anterior pituitary during organogenesis and are distinguished by pro-opiomelanocortin (POMC) gene expression. Earlier studies in our laboratory defined three DNA regions (sites 1, 2 and 3) within promoter sequences at the 5′-end of the rat POMC gene (-323/-34) that cooperatively targeted cell-specific gene expression to corticotrophs and melanotrophs in transgenic mice. In this study we analysed the DNA-nuclear protein interactions underlying this functional activity. We demonstrated that the transcriptional activator SP1 interacts with GC-rich regions in sites 1 (-146/-136) and 2 (-201/-192) and an unidentified protein, which we call PP1 (putative pituitary POMC1), interacts with AT-rich regions in sites 2 (-202/-193) and 3 (-262/-253). The PP1-binding activity appears to be specific to cells that express the POMC gene because it was detected in nuclear extracts prepared from AtT20 corticotroph cells and mouse melanotroph tumours but not from GH4 pituitary tumour cells, HeLa cells or liver. Site-directed mutagenesis of core binding sequences demonstrated that PP1 is required for the correct cell-specific expression of the POMC gene in the pituitary gland of transgenic mice and SP1 appears to support such an expression. The best core binding sequence for PP1 is TAAT, a possible transcription factor homeodomain contact site. However, PP1 is distinct from Brn 3.0, a POU protein that also binds to site 3. We conclude that PP1 is a transcriptional activator for pituitary-specific POMC gene expression.

The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5121-5124 ◽  
Author(s):  
Thomas Bee ◽  
Emma L.K. Ashley ◽  
Sorrel R.B. Bickley ◽  
Andrew Jarratt ◽  
Pik-Shan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Reporter Gene ◽  
Specific Pattern ◽  
Reporter Gene Expression ◽  
Alternative Promoters ◽  
Specific Expression ◽  
Hematopoietic Stem

Abstract The transcription factor Runx1 plays a pivotal role in hematopoietic stem cell (HSC) emergence, and studies into its transcriptional regulation should give insight into the critical steps of HSC specification. Recently, we identified the Runx1 +23 enhancer that targets reporter gene expression to the first emerging HSCs of the mouse embryo when linked to the heterologous hsp68 promoter. Endogenous Runx1 is transcribed from 2 alternative promoters, P1 and P2. Here, we examined the in vivo cis-regulatory potential of these alternative promoters and asked whether they act with and contribute to the spatiotemporal specific expression of the Runx1 +23 enhancer. Our results firmly establish that, in contrast to zebrafish runx1, mouse Runx1 promoter sequences do not confer any hematopoietic specificity in transgenic embryos. Yet, both mouse promoters act with the +23 enhancer to drive reporter gene expression to sites of HSC emergence and colonization, in a +23-specific pattern.

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