A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

Development ◽  
1991 ◽  
Vol 112 (3) ◽  
pp. 807-811 ◽  
Author(s):  
K. Schughart ◽  
C.J. Bieberich ◽  
R. Eid ◽  
F.H. Ruddle
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Developmental Stages ◽  
Regulatory Region ◽  
Homeobox Gene ◽  
Regulatory Elements ◽  
Body Region ◽  
Lacz Gene ◽  
Mouse Lines

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.

scholarly journals Renal Anemia Model Mouse Established by Transgenic Rescue with an Erythropoietin Gene Lacking Kidney-Specific Regulatory Elements

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Ikuo Hirano ◽  
Norio Suzuki ◽  
Shun Yamazaki ◽  
Hiroki Sekine ◽  
Naoko Minegishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Renal Anemia ◽  
Content Type ◽  
Transgenic Expression ◽  
Complex Process ◽  
Model Mouse ◽  
Mouse Lines

ABSTRACT The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3′ region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal E po regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

scholarly journals −245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2326-2333 ◽  
Author(s):  
Zheng Cui ◽  
Michael P. Reilly ◽  
Saul Surrey ◽  
Elias Schwartz ◽  
Steven E. McKenzie
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
Reporter Gene Expression ◽  
Platelet Factor ◽  
Flanking Region ◽  
Mouse Lines

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

scholarly journals −245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2326-2333 ◽  
Author(s):  
Zheng Cui ◽  
Michael P. Reilly ◽  
Saul Surrey ◽  
Elias Schwartz ◽  
Steven E. McKenzie
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
Reporter Gene Expression ◽  
Platelet Factor ◽  
Flanking Region ◽  
Mouse Lines

Abstract Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

scholarly journals Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3442-3449 ◽  
Author(s):  
M S Donoviel ◽  
N Kacherovsky ◽  
E T Young
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Reporter Gene ◽  
Copy Number ◽  
Glucose Repression ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

scholarly journals Response of the 5′-flanking region of the human 25-hydroxyvitamin D 1α-hydroxylase gene to physiological stimuli using a transgenic mouse model

2005 ◽  
Vol 34 (1) ◽  
pp. 237-245 ◽  
Author(s):  
I Hendrix ◽  
P H Anderson ◽  
J L Omdahl ◽  
B K May ◽  
H A Morris
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Vitamin D Deficiency ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Dietary Calcium ◽  
Mrna Levels ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
Flanking Region

The enzyme 25-hydroxyvitamin D 1α-hydroxylase, or CYP27B1, is the key enzyme in the two-step activation process of vitamin D to 1,25-dihydroxyvitamin D (1,25D). While a number of regulators of the renal CYP27B1 enzyme activity have been recognized for some years, their underlying molecular mechanisms remain largely unknown, and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated. We have generated a transgenic mouse line that expresses 1501 bp of 5′ flanking region together with 44 bp of 5′ untranslated region of the human CYP27B1 gene fused to the firefly luciferase reporter gene. Animals expressing the luciferase gene demonstrated that both luciferase protein and mRNA for CYP27B1 were localized to proximal convoluted tubule cells of the kidney. In 2-week-old animals, the expression of the transgene and the endogenous CYP27B1 mRNA levels in the kidney were highest and fell with increasing age. Both reporter gene expression and CYP27B1 mRNA levels were downregulated in response to increasing amounts of dietary calcium in a dose-dependent manner. Vitamin D deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. Interestingly, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting either that there is a post-transcriptional mechanism that increases the amount of CYP27B1 mRNA or that other regulatory elements are required to maximize the effect of vitamin D deficiency. These findings demonstrate that the 1501 bp 5′ flanking region of the CYP27B1 gene directs expression to the proximal convoluted tubules of the kidney and is responsible for increasing transcriptional activity when dietary calcium and vitamin D levels are depleted. It also responds in the kidney to the physiological regulators of development and ageing.

Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Development ◽  
2001 ◽  
Vol 128 (12) ◽  
pp. 2341-2350
Author(s):  
Makoto Kobayashi ◽  
Keizo Nishikawa ◽  
Masayuki Yamamoto
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Regulatory Region ◽  
Ectopic Expression ◽  
Transgenic Fish ◽  
Functional Domain ◽  
Cis Acting ◽  
Gfp Reporter ◽  
Gata Motif

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.

Targeted modification of promoters

2021 ◽  
pp. 247-278
Author(s):  
Andika Gunadi ◽  
◽  
Ning Zhang ◽  
John J. Finer ◽  
◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Promoter Region ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Upstream Regulatory Region ◽  
Precise Control ◽  
Coding Regions ◽  
Gene Coding ◽  
Altered Regulation

Although most genome editing efforts focus on modifications to gene coding regions, this chapter emphasizes genome editing of the upstream regulatory regions. Thoughtful editing of the promoter region will ultimately lead to improved plants, modified for more precise control of the intensity and specificity of native gene expression. In this chapter, we present an overview of the promoter or upstream regulatory region of a gene, and describe how this sequence is defined and studied. We then describe how the composition and arrangements of cis-regulatory elements within the promoter and the leading intron associated with the promoter region have been studied using classical transgenic approaches to reveal what regulatory components might be suitable for genome editing approaches. Finally, we offer some suggestions for pursuit of promoter editing and gene expression modulation, which will eventually lead to modified plants with an altered regulation of native gene expression.

scholarly journals RETRACTED ARTICLE: The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lin Zhang ◽  
Zhiqiang Song ◽  
Fangfang Li ◽  
Xixi Li ◽  
Haikun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Drought Stress ◽  
Reporter Gene ◽  
Binding Sites ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Cis Elements ◽  
Gus Reporter ◽  
Almost All

Abstract Background Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. Results In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5′ deletion mutants of these promoters, it was determined that TaGAPCp2P (− 1312~ − 528) and TaGAPCp3P (− 2049~ − 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (− 1197~ − 635) and TaGAPCp3P (− 1456~ − 1144 and − 718~ − 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (− 1135 and − 985) and TaGAPCp3P (− 1414 and − 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. Conclusion The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice

2003 ◽  
Vol 284 (3) ◽  
pp. L481-L488 ◽  
Author(s):  
Li Yang ◽  
Angela Naltner ◽  
Allison Kreiner ◽  
Dong Yan ◽  
Angelynn Cowen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Lung Development ◽  
Alveolar Type ◽  
Transcriptional Level ◽  
Lacz Gene ◽  
Type Ii ◽  
Enhancer Region

Regulation of the surfactant protein B gene (SP-B) is developmentally controlled and highly tissue specific. To elucidate the SP-B gene temporal/spatial expression pattern in lung development at the transcriptional level, a transgenic mouse model line carrying the human SP-B (hSP-B) 1.5-kb 5′-flanking regulatory region and the lacZ gene was established. Expression of hSP-B 1.5-kb lacZ gene started at the onset of lung formation [embryonic day 9 (E9)] and was restricted to epithelial cells throughout prenatal and postnatal lung development. In the adult lung, hSP-B 1.5-kb lacZ gene expression was restricted to bronchiolar and alveolar type II epithelial cells. In lung explant culturing studies, the hSP-B 1.5-kb lacZ gene was highly expressed in newly formed epithelial tubules during the respiratory branching process. In a second transgenic mouse line, an enhancer region, which binds to thyroid transcription factor-1, retinoic acid receptor, signal transducers and activators of transcription 3, and nuclear receptor coactivators (SRC-1, ACTR, TIF2, and CBP/p300), was deleted from the hSP-B 1.5-kb lacZ gene. The deletion abolished hSP-B lacZ gene expression in bronchiolar epithelial cells and significantly reduced its expression level in alveolar type II epithelial cells in transgenic mice.

scholarly journals Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver

1996 ◽  
Vol 314 (3) ◽  
pp. 903-909 ◽  
Author(s):  
Franck RENCUREL ◽  
Gérard WAEBER ◽  
Bénédicte ANTOINE ◽  
Francis ROCCHICCIOLI ◽  
Paulette MAULARD ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Reporter Gene ◽  
Glucose Transporter ◽  
Regulatory Region ◽  
Proximal Fragment ◽  
Mrna Accumulation ◽  
Glut2 Gene

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5´ regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase Km 16 mM; hexokinase I Km 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

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