Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

2002 ◽  
Vol 283 (4) ◽  
pp. H1439-H1445 ◽  
Author(s):  
Margot C. LaPointe ◽  
Xiao-Ping Yang ◽  
Oscar A. Carretero ◽  
Quan He
Keyword(s):  
Reporter Gene ◽  
Cardiac Myocyte ◽  
Luciferase Activity ◽  
Recombinant Adenovirus ◽  
Left Ventricular ◽  
Luciferase Reporter ◽  
Mouse Heart ◽  
Free Wall

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1β, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

Abstract 96: Inactivation of Neddylation Causes Left Ventricular Noncompaction Cardiomyopathy Through Suppressing YAP Signaling

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Jianqiu Zou ◽  
Wenxia Ma ◽  
Jie Li ◽  
Rodney Littlejohn ◽  
Il-man Kim ◽  
...  
Keyword(s):  
Cardiac Development ◽  
Heart Diseases ◽  
Left Ventricular ◽  
Late Gestation ◽  
Mouse Heart ◽  
Cardiomyocyte Proliferation ◽  
Transcription Cofactor ◽  
Wild Type Counterpart

Rationale: Cardiac development is orchestrated by a number of growth factors, transcription factors and epigenetic regulators, perturbation of which can lead to congenital heart diseases and cardiomyopathies. However, the role of novel ubiquitin-like protein modifiers, such as NEDD8 (neural precursor cells expressed developmentally downregulated 8), in cardiac development is unknown. Objectives: The objective of this study was to determine the significance of NEDD8 modification (neddylation) during perinatal cardiac development. Methods and Results: Neddylated proteins and NEDD8 enzymes were highly abundant in fetal and neonatal hearts but downregulated in adult hearts. We employed an αMHC Cre transgene to delete NAE1, a subunit of the NEDD8 E1 enzyme, in the perinatal mouse heart. Cardiac-specific deletion of NAE1 (NAE1 CKO ) significantly decreased neddylated proteins in the heart. The NAE1 CKO mice displayed cardiac hypoplasia, ventricular non-compaction and heart failure during late gestation, which became more pronounced by postnatal day 1 and led to perinatal lethality. Mechanistically, genetic deletion or pharmacological inhibition of NAE1 resulted in accumulation of Hippo kinases Mst1 and LATS1/2, which in turn phosphorylated and inactivated YAP, a transcription cofactor necessary for cardiomyocyte proliferation, leading to dysregulation of a number of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro . Reactivation of YAP signaling by overexpression of a constitutively-active YAP mutant (YAP 5SA ), but not its wild-type counterpart, overcame the blockade of cardiomyocyte proliferation induced by inhibition of NAE1. Conclusions: Our findings establish the importance of neddylation in the heart, more specifically, in ventricular chamber maturation, and identify neddylation as a novel regulator of Hippo-YAP signaling to promote cardiomyocyte proliferation.

scholarly journals Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

2014 ◽  
Vol 2014 ◽  
pp. 1-11
Author(s):  
Ya-Ju Hsieh ◽  
Luen Hwu ◽  
Chien-Chih Ke ◽  
Skye Hsin-Hsien Yeh ◽  
Chien-Feng Lin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Rational Design ◽  
Multimodality Imaging ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Fluorescence Signal ◽  
Red Fluorescence ◽  
Simplex Virus

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase(fl), monomeric red fluorescence protein(mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant(ttksr39)were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro andin vivowere determined by luciferase reporter assay,H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels ofH-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak forin vivoimaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter constructDsRedm-fl-ttksr39for more effective and sensitivein vivoanimal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

scholarly journals Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice

2004 ◽  
Vol 32 (3) ◽  
pp. 689-701 ◽  
Author(s):  
JG Lemmen ◽  
RJ Arends ◽  
AL van Boxtel ◽  
PT van der Saag ◽  
B van der Burg
Keyword(s):  
Reporter Gene ◽  
Imaging System ◽  
Luciferase Activity ◽  
Estrogenic Activity ◽  
Receptor Activation ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
In Vivo Model ◽  
Luciferase Reporter Gene

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

scholarly journals Identification of a novel distal enhancer in human adiponectin gene

2008 ◽  
Vol 200 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Katsumori Segawa ◽  
Morihiro Matsuda ◽  
Atsunori Fukuhara ◽  
Kentaro Morita ◽  
Yosuke Okuno ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Luciferase Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Adiponectin Gene ◽  
Distal Enhancer

Adiponectin is exclusively expressed in adipose tissue and secreted from adipocytes, and shows anti-diabetic and anti-atherogenic properties. However, the precise transcriptional mechanism of adiponectin remains elusive. In this study, the 5′ flanking promoter region of human adiponectin gene was analyzed using UCSC genome browser, and a 10 390-bp fragment, containing an evolutionally conserved region among species, was investigated. The luciferase reporter assay using this fragment identified a novel distal enhancer of human adiponectin gene. Promoter constructs with the distal enhancer exhibited high promoter activities in 3T3-L1 mature adipocytes. However, no such activity was observed in other types of cell lines. The distal enhancer is highly conserved, and contains two completely conserved CCAAT boxes. In 3T3-L1 mature adipocytes, deletion or each point mutation of these CCAAT boxes markedly reduced luciferase activity driven by adiponectin promoter. Knockdown of CCAAT/enhancer-binding protein α (CEBPA; also known as C/EBPα) using small interfering RNA diminished adiponectin mRNA expression and luciferase activity driven by adiponectin promoter with the distal enhancer. However, adiponectin promoter with each mutation of two CCAAT boxes in the distal enhancer did not respond to knockdown of CEBPA expression. Furthermore, CEBPA bound to the distal enhancer both in vitro and in vivo. We also identified a proximal promoter region responsible for transcriptional activation by the distal enhancer in human adiponectin gene. Our results indicate that CEBPA plays a pivotal role in the transcription of human adiponectin gene via the distal enhancer and proximal region in its promoter.

scholarly journals Pharmacological priming of adipose-derived stem cells promotes myocardial repair

2016 ◽  
Vol 64 (1) ◽  
pp. 50-62 ◽  
Author(s):  
Jana S Burchfield ◽  
Ashley L Paul ◽  
Vishy Lanka ◽  
Wei Tan ◽  
Yongli Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cardiac Function ◽  
Functional Recovery ◽  
Cardiac Myocyte ◽  
Left Ventricular ◽  
Adipose Derived Stem Cells ◽  
Myocardial Repair ◽  
Myocyte Differentiation

Adipose-derived stem cells (ADSCs) have myocardial regeneration potential, and transplantation of these cells following myocardial infarction (MI) in animal models leads to modest improvements in cardiac function. We hypothesized that pharmacological priming of pre-transplanted ADSCs would further improve left ventricular functional recovery after MI. We previously identified a compound from a family of 3,5-disubstituted isoxazoles, ISX1, capable of activating an Nkx2-5-driven promoter construct. Here, using ADSCs, we found that ISX1 (20 mM, 4 days) triggered a robust, dose-dependent, fourfold increase in Nkx2-5 expression, an early marker of cardiac myocyte differentiation and increased ADSC viability in vitro. Co-culturing neonatal cardiomyocytes with ISX1-treated ADSCs increased early and late cardiac gene expression. Whereas ISX1 promoted ADSC differentiation toward a cardiogenic lineage, it did not elicit their complete differentiation or their differentiation into mature adipocytes, osteoblasts, or chondrocytes, suggesting that re-programming is cardiomyocyte specific. Cardiac transplantation of ADSCs improved left ventricular functional recovery following MI, a response which was significantly augmented by transplantation of ISX1- pretreated cells. Moreover, ISX1-treated and transplanted ADSCs engrafted and were detectable in the myocardium 3 weeks following MI, albeit at relatively small numbers. ISX1 treatment increased histone acetyltransferase (HAT) activity in ADSCs, which was associated with histone 3 and histone 4 acetylation. Finally, hearts transplanted with ISX1-treated ADSCs manifested significant increases in neovascularization, which may account for the improved cardiac function. These findings suggest that a strategy of drug-facilitated initiation of myocyte differentiation enhances exogenously transplanted ADSC persistence in vivo, and consequent tissue neovascularization, to improve cardiac function.

scholarly journals MiR-20b Down-Regulates Intestinal Ferroportin Expression In Vitro and In Vivo

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1135 ◽  
Author(s):  
Shuxia Jiang ◽  
Xi Fang ◽  
Mingni Liu ◽  
Yingdong Ni ◽  
Wenqiang Ma ◽  
...  
Keyword(s):  
Protein Expression ◽  
Luciferase Activity ◽  
Family Members ◽  
Luciferase Reporter ◽  
Iron Level ◽  
Cellular Iron ◽  
Reporter Assays ◽  
Post Transcriptional Regulation

Ferroportin (FPN) is the only known cellular iron exporter in mammalian. However, post-transcriptional regulation of intestinal FPN has not yet been completely understood. In this study, bioinformatics algorithms (TargetScan, PicTar, PITA, and miRanda) were applied to predict, screen and obtain microRNA-17 family members (miR-17, miR-20a, miR-20b, and miR-106a) targeting FPN, ‘seed sequence’ and responding binding sites on the 3′untranslated region (3′UTR) region of FPN. Dual-luciferase reporter assays revealed miRNA-17 family members’ mimics decreased the luciferase activity, whereas their inhibitors increased the luciferase activity. Compared with the FPN 3′UTR wild type reporter, co-transfection of a miRNA-17 family members’ over-expression plasmids and FPN 3′UTR mutant reporters enhanced the luciferase activity in HCT116 cells. Transfection with miR-20b overexpression plasmid significantly enhanced its expression, and it inhibited endogenous FPN protein expression in Caco-2 cells. Additionally, tail-vein injection of miR-20b resulted in increasing duodenal miR-20b expression, decreasing duodenal FPN protein expression, which was closely related to lower plasma iron level in mice. Taken together, these data suggest that the miR-20b is identified to regulate intestinal FPN expression in vitro and in vivo, which will provide a potential target for intestinal iron exportation.

Inducible regulation of human brain natriuretic peptide promoter in transgenic mice

2001 ◽  
Vol 280 (1) ◽  
pp. H368-H376 ◽  
Author(s):  
Quan He ◽  
Ding Wang ◽  
Xiao-Ping Yang ◽  
Oscar A. Carretero ◽  
Margot C. LaPointe
Keyword(s):  
Transgenic Mice ◽  
Brain Natriuretic Peptide ◽  
Cardiac Myocytes ◽  
Natriuretic Peptide ◽  
Luciferase Activity ◽  
Left Ventricular ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Coronary Artery Ligation

Studies have shown that brain natriuretic peptide (BNP) gene expression is rapidly induced in the infarcted heart and that plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous in vitro work using transiently transfected neonatal rat cardiac myocytes has shown that the human BNP (hBNP) promoter, in particular a region extending from −127 to −40 relative to the start site of transcription, is more active in cardiac myocytes than in fibroblasts. To study tissue-specific and transcriptional regulation of the hBNP gene in vivo, we generated transgenic mice containing the proximal hBNP promoter (−408 to +100) coupled to a luciferase reporter gene. In four lines of transgenic mice, luciferase activity was ∼33- to 100-fold higher in the heart than in other tissues, including the whole brain. To test whether the transgene responded to a pathophysiological stimulus, we induced infarction by coronary artery ligation. Luciferase activity was fivefold higher in the infarcted region of the left ventricle at 48 h than in sham-operated animals and remained elevated for 4 wk. Endogenous BNP mRNA was similarly increased in the infarcted hearts of a separate group of mice. We conclude that 1) the proximal 408-bp region of the hBNP promoter confers cardiac-specific expression and 2) myocardial infarction activates the proximal hBNP promoter in vivo. These data suggest that we have a valid model for the study of basal and inducible regulation of the hBNP gene in vivo.

scholarly journals Hsa_circ_0011385 knockdown represses cell proliferation in hepatocellular carcinoma

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Chuangye Ni ◽  
Shikun Yang ◽  
Yang Ji ◽  
Yunfei Duan ◽  
Wenjie Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

AbstractCircular RNAs (circRNAs), continuous loops of single-stranded RNA, regulate gene expression during the development of various cancers. However, the function of circRNAs in hepatocellular carcinoma (HCC) is rarely discussed. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA levels of circ_0011385, miR-361-3p, and STC2 in 96 pairs of HCC tissues (tumor tissues and adjacent normal tissues), HCC cell lines, and L02 (human normal liver cell line) cells. The relationships between circ_0011385 expression and clinical features of HCC were evaluated. Functional experiments in vitro or in vivo were used to evaluate the biological function of circ_0011385. Bioinformatics analysis was performed to predict miRNAs and mRNAs sponged by circ_0011385. RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assays were used to elucidate the interactions among circ_0011385, miR-361-3p, and STC2 (stanniocalcin 2). ChIP and dual-luciferase reporter gene assays were used to identify the upstream regulator of circ_0011385. High expression of circ_0011385 was observed in HCC tissues and cell lines and was significantly associated with tumor size, TNM stage, and prognosis. In addition, inhibition of circ_0011385 expression prevented the proliferation of HCC cells in vitro and in vivo. Circ_0011385 sponged miR-361-3p, thereby regulating the mRNA expression of STC2. In addition, the transcription of circ_0011385 was regulated by SP3. Circ_0011385 knockdown suppressed cell proliferation and tumor activity in HCC. Circ_0011385 may therefore serve as a new biomarker in the diagnosis and treatment of HCC.

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