Instrument Grant Program Recipients

Congratulations to all of our grant recipients. We look forward to receiving your application and adding your name to this list of grantees.

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Microplate Luminometer
Pamela Kreeger
Assistant Professor
Dept. of Biomedical Engineering
University of Wisconsin-Madison
kreeger.openwetware.org
kreeger@wisc.edu

My lab is interested in how outside signals such as hormones and growth factors are interpreted in the cellular signaling network. We use a variety of high-throughput experimental methods to gather information about multiple signaling pathways and  perform computational analysis to identify new drug targets.  We use the Veritas Microplate Luminometer with Dual Injectors for many of our experimental assays due to the sensitivity of luminescent assays combined with the high-throughput design.  In particular, we rely on reporter gene assays to determine the activity of transcription factors, luminescent assays for kinase activity and cAMP levels to monitor signaling activity, and a variety of luminescent assays to characterize cell behaviors such as apoptosis and proliferation.

Microplate Multimode Reader set up as a Luminometer
Michelle Duffourc
Director, Asst. Professor
East Tennessee State University, Molecular Biology Core Facility
Johnson City, TN

I am the Director of my university’s Molecular Biology Core Facility (MBCF), a multi-user facility dedicated to advancing molecular biology-based research and education for all interested students and personnel. The MBCF accomplishes this mission by 1) providing access to, and training in, the theory and use of instrumentation not affordable by smaller laboratories; 2) providing consultation services and technical assistance to educators and researchers; 3) performing analyses on a fee-for-service basis; and 4) offering a variety of educational experiences including classroom demonstrations, student research rotations and hands-on intensive workshops. Since the MBCF is the ONLY facility of its kind within a 90 mile radius all faculty, staff, and students (undergraduate, graduate, or medical) interested in biotechnology utilize the programs offered by the MBCF either directly or indirectly (e.g., a faculty member receiving training and bringing it to their laboratory). Further, while the MBCF was clearly developed to serve the needs of the East Tennessee State University community, it routinely hosts faculty and students from nearby colleges so that they may have access to technology not available to them otherwise.

Microplate Multimode Reader set up as a Luminometer
(June 2007 – This is our first grant recipient of this instrument)

Tanja Stoyan, Ph.D. Coordinator Undergraduate Teaching Laboratory
University of California Santa Barbara
Santa Barbara, California

I coordinate and teach laboratory classes for undergraduates in pharmacology and immunology. Approximately 60 students graduate from these classes each year with a B.S. in Pharmacology or  a B.S. in Microbiology/Cell Biology. These are classes for seniors and many of our students go on to work in the Biotech and Pharmaceutical Industries after they graduate. We are intending to expose our students to a wide variey of biochemical techniques that apply to molecular pharmacology. Recently we have developed new exercises for our classes using Flow Cytometry. My goal is to introduce our Pharmacology and Immunology students to the field of Luminometry and to develop several exercises that use this powerful technique. I will give you a brief overview about the Pharmacology program. The lab courses require pharmacology majors to spend one day each week for two quarters performing experiments to demonstrate the site and mechanism of action of a wide variety of drugs and chemical agents using different in vitro isolated tissue preparations, as well as in vivo, using anesthetized mice and rabbits. The winter quarter lab is designed to teach common biochemical techniques that are routinely used to investigate site and mechanism of action of drugs. The series of laboratory exercises include an ELISA assay to determine the concentration of protein in a sample, a radioactive drug-receptor-binding filter assay, a pharmacokinetic study to identify metabolites following different routes of administration of a therapeutic agent, isolation of liver microsomes for drug metabolism study, including identification of p450 enzymes by gel electrophoresis and immuno-blot, the amplification of G protein cDNA using PCR and a new Flow Cytometry exercise to determine the EC50 of drugs on apoptotic Jurkat cells. In addition to the experiments described above the students are required to complete a 3-dimensional molecular modeling computer exercise teaching them how to interpret structure-activity relationship of drugs and molecular binding at drug-receptor sites. The students use various software packages to analyze their data (Excel, Sigmaplot, Flow Cytometry software). Our goal is to keep the class up to date with new technologies and instrumentation. We feel that luminometry is a very important research tool. We will be using Cell Viability and Apoptosis assays, Cell titer glo, Caspase-Glo, High Through put compound screening: p450 glo, kinase-glo plus assay, pGL4, luciferase reporter vectors, Dual-glo luciferase assay system. One assay that we are interested in developing is an assay to screen for GPRC modulators which is described in Cell Notes, Issue 16, 2006 from Promega. We would introduce a luciferase reporter plasmid into various cell lines and have the students add drugs from an 80 compound library to the cells (alternatively a series of dilutions of one single drug). We would then assay for firefly and Renilla luciferase activity using the Dual-glo assay system. The results would be measured in a 96 microplate luminometer. Another application would be to assay apoptotic activity of drugs in our compound library on Jurkat cells with a luminescent assay. A high throughput screen using Flow cytometry has been difficult to do in the class format since the time for reading the 96 well plate is too long.

(2008) 96-well Luminometer (Granted once per year in December)

Valerie Hu
Professor of Biochemistry and Molecular Biology
The George Washington University Medical Center
Biochemistry and Molecular Biology
Washington, DC USA

Personalized Diagnosis and Treatment of Autism Spectrum Disorders
The goals of my research program are:
1) To identify expressed biomarkers of autism spectrum disorders (ASD) for molecular screening and diagnosis;
2) To understand the pathobiology of ASD in order to design drugs specific to the
defect or deficiency;
3) To develop a method for classification of cases according to “biological phenotype” or severity in order to identify subgroups that may benefit from specific medications or therapies.
Summary of research findings towards accomplishment of these goals:
1) We have clustered individuals with ASD according to behavioral and functional phenotypes;
2) We have utilized DNA microarrays to associate distinct gene expression profiles with each of 3 phenotypes of ASD;
3) The most severe phenotype of ASD is associated with dysregulation of circadian rhythm genes, confirmed by quantitative RT-PCR analyses.

(2007) 96-well Luminometer (Granted once per year in December)
Dr. Brandon White
San Jose State University
I am interested in transcriptional regulation of the Notch signalling system as it relates to development, adult, and cancer regulation. I primarily use the luminometer with
Promega's dual reporter assay and will be starting some apoptosis assays
with Promega's newest reporter system. I want to be able to perform these
assays in a 96-well format for high-thorougput screening.

(2006) 96-well Luminometer (Granted once per year in December)
Adam Carroll, Assistant Adjunct Professor
University of California San Francisco
Biochemistry & Biophysics Department
San Francisco, California
http://derisilab.ucsf.edu/core
I run a shared instrumentation facility with a focus on training rather than on service.  My research goals are aligned with the users of the facility, who bring a wide variety of topics into the lab.  We have people working on projects ranging from bacterial to viral to human, doing different things from the most basic science to drug screening.  Our goal is to ensure that the scientists at UCSF, as well as other UC institutions, have access to advanced instrumentation for high-throughput biology, whatever the specific topic of research. We have a number of high-throughput liquid handlers, so many projects could be applied to a Veritas luminometer.    We have researchers interested in established luciferase protocols, but also expect users of the facility to develop new assays with the tools we provide. My laboratory is the ideal place for a Turner Luminometer to get the maximum possible exposure to the largest number of researchers.  We have several other small instruments that we have received through university grant programs; the granting companies have been very pleased with the results.  As a place of interactions on the topic of new technologies for research at UCSF, my lab is unparalleled.

(2005) 96-well Luminometer
Dr. Wolfgang Leidtke of Duke University was granted a Veritas Microplate Luminometer. In his laboratory, he is trying to understand transcriptional regulation of genes in the nervous system that are involved in signal transduction in response to osmotic and mechanical stimuli.  High-throughput reporter assays with pharmacologically active inhibitors of signaling pathways will be conducted with 2 kB 5' sequences of such genes driving luciferase reporter constructs.

(2004) 96-well Luminometer
Dr. Karin Borgh of the Biopharmaceutical Technology Center Institute (BTCI) in Madison, Wisconsin, was granted the first Veritas Microplate Luminometer. BTCI is a non-profit educational organization providing courses for scientific personnel in academia and industry. The courses are offered to academic researchers, teaching faculty, postdoctoral candidates, graduate and undergraduate students, industry employees and high school students. They will be working with assays like SNP (READIT), CaspACE, Kinase, functional protein capture and analysis as well as cell viability.

(2008) Single Tube Multimode Reader set up as a Luminometer
Kathleen Tallman
Associate Professor
Azusa Pacific University
Department of Biology and Chemistry
Azusa, CA USA

The Biology and Chemistry department at Azusa Pacific University has a strong history in excellent teaching and preparation of undergraduate students for careers in the health sciences, biomedical research, and other areas of biology. Our students have a high acceptance rate to medical school and doctoral programs in top universities for biomedical research. The Biology and Chemistry department offers undergraduate degrees in biology, biochemistry, chemistry, and applied health; however, the university offers several graduate degrees including doctoral programs in nursing and physical therapy. To continue a top level of preparation, the university is nearing completion of a newly constructed science building which will be occupied in May of 2009.

(2008) Single Tube Multimode Reader set up as a Luminometer

Hexin Chen, Ph.D.
Assistant Professor
University of South Carolina
Biological Sciences Department
Columbia, SC USA

I am an assistant tenure track biology professor at the University of South Carolina. I am pursuing both basic and translational research in my laboratory. My basic research focuses on the role of HOX gens in breast cancer development. HOX genes encode a large family of transcriptional regulatory proteins containing a conserved DNA binding domain called homeodomain. Homeodomain-containing proteins determine the phenotypic outcome of a variety of embryonic cells by ensuring that specifically timed differentiation programs are set in place in these cells. In recent years, a large body of literature has been generated about the abnormal expression of HOX genes in a variety of tumors. However, very little is known about how abnormal expression of HOX genes can lead to tumor formation. Our previous studies have shown that HOXA5 acts as a tumor suppressor gene, on the other hand, HOXB7 acts as an oncogene. As a classical transcription factor, we hypothesize that HOXA5 and HOXB7 exert their functions by directly or indirectly regulating the expression of their target genes. Microarray and western blot analysis have identified many potential target genes of HOXA5 and HOXB7 (Chen et al. 2005.
Identification of transcriptional targets of HOXA5. J. Bio. Chem. 280:19373-80). In order to identify their direct targets, we will clone the promoters of selected candidate genes by PCR and insert into our modified luciferase-reporter vector. The response of these promoters to HOXA5 and HOXB7expression will be studied by transient transfection assays using Promega dual luciferase kit. My translational research project is to determine whether immunological response can predict the clinical outcome of Herceptin (HER-2/neu monoclonal antibody) -treated breast cancer patient. Antitumor monoclonal antibodies (mAb) represent a major advance in the therapy of cancer. In the treatment of HER-2+ breast cancer, the HER-2/neu monoclonal antibody Herceptin in combination with chemotherapy increases survival in the metastatic setting and enhances disease-free survival in the adjuvant
setting. But unfortunately only 30-60% of patients may benefit from Herceptin-mediated treatment. Antitumor antibodies, as opposed to protein tyrosine kinase inhibitors, contain Fc domains that may also mechanistically contribute via the induction of an innate and/or adaptive antitumor immune response. We hypothesized that tumor responses in patients treated with combination chemotherapy and Herceptin would be accompanied by alterations in antitumor immunity, which may serve as biomarker to predict the clinical outcome of Herceptin-mediated treatment in these patients. We will measure global immune response using an in vitro assay (CytexTM Immune Cell Function Assay), which was designed to measure the concentration of ATP from CD4 cells following stimulation with phytohemagglutinin. This measurement is made on heparin anti-coagulated whole blood using a luminometer and luciferin/luciferase (http://www.cylex.net/products.html). The luminometer which can be used to measure both luciferase and ATP levels would be extremely helpful to our research.

Single Tube Multimode Reader set up as a Luminometer
Kristen Wright, Graduate Student
The George Washington University, Pharmacology and Physiology
Washington, DC

Our research goal is to elucidate the molecular mechanisms involved in survival signaling and carcinogenesis after genotoxic insult. Upon genotoxin exposure, damaged cells will undergo a transient checkpoint arrest resulting in either apoptosis or terminal growth arrest; however, a small group of cells may survive. We are interested in the ability of a cell to survive genotoxin exposure, and we postulate that the clonogenic survivors may be predisposed to neoplastic progression through genetic or epigenetic alterations. Mitochondria are critical to homeostatic control, and are at the crux of cell growth and death. Hallmarks of neoplastic progression include limitless replicative potential and evasion of apoptosis. We propose that the selection of cells with mitochondrial dysregulation may lead to death resistance. The enzyme hexokinase (HK) is responsible for the first
enzymatic step in glycolysis. The HK II isoform is known to specifically bind to the mitochondria and is thought to couple glycolysis to oxidative phosphorylation. Many cancer cells are known to increase glycolysis to generate ATP for energy as they become more death resistant, even in the presence of oxygen. This phenomenon, known as the “Warburg effect,” is due to mitochondrial respiratory defects, which is characteristic of malignant transformation. We have data that implies a connection between mitochondrial associated-HKII and mitochondrial energy metabolism in a
cellular model of death resistance; therefore, we plan to investigate the bioenergetic signature of these cells by measuring intracellular ATP/ADP levels basally and upon genotoxin exposure. Moreover, we hope to elucidate a mechanism of mitochondrial regulation in survival signaling and death resistance, which may play a role in neoplastic progression. We will primarily measure ATP and ADP via a bioluminescence assay using recombinant firefly luciferase and D-luciferin.

Single Tube Multimode Reader set up as a Luminometer
Radhika Andavolu, Director
Genetics Research Institute of the Desert
Rancho Mirage, California
www.gridonline.org
Microarray technology to define molecular signatures of prostate tumors that will provide additional predictive value over conventional markers of outcome and be useful in helping to guide clinical management. The assays I am running are Panomics Quantigene assays for Gene Expression in Tumor and normal tissues.

Single Tube Multimode Reader set up as a Luminometer
David Salvay, Graduate Student (MD/PhD Candidate)
Northwestern University
Institute for Bionanotechnology in Medicine
Chicago, Illinois
http://www.ibnam.northwestern.edu/
Our research focuses on how microporous, biodegradable tissue engineering scaffolds may be used as platforms for gene delivery. Our goal is to develop devices capable of being tuned to provide gene expression of varying durations and levels for the treatment of a variety of clinically relevant conditions. These range from expressing Factor VIII or IX for the treatment of hemophilia to creating local regions of immunosuppression to support allo- or xenogenic cell transplantation.
List of Assays = Luminescence, absorbance and fluorescence
Luminescence to quantify luciferase expression from in vitro transfection studies and in vivo gene expression following scaffold implantation from tissue homogenates. Absorbance to assess protein concentration (BCA). Fluorescence for nucleic acid quantification (PicoGreen and Hoechst) to assess the release kinetics of DNA from our scaffolds. This machine would be extremely helpful to our research and enable us to perform a number of assays we are currently unable to perform on-site.

Single Tube Multimode Reader set up as a Luminometer
Dr. Raju C. Reddy is an Assistant Professor of Internal Medicine in the Division of Pulmonary and Critical Care Medicine at the University of Michigan School of Medicine. He was granted a Modulus Luminometer to study the role of the nuclear hormone receptor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), in lung tumorigenesis, pulmonary fibroproliferative responses, and alveolar macrophage function. Further characterizing the functional significance of PPAR-gamma in pulmonary (patho)biology will potentially result in the identification of novel therapeutic targets.

Single Tube Luminometer
Herman Lehman, Associate Professor and Chair
Hamilton College, Department of Biology
Clinton, NY USA https://my.hamilton.edu/academics/department.html?dept=Biology

Research Goals = Further Characterization of Interferon Responsive Factor 2 Binding Protein 1 as a novel cardiac-enriched transcription cofactor that coactivates the vascular endothelial growth factor promoter.

My laboratory is broadly interested in the regulation of neurotransmitters and growth factors. This year, a student in my laboratory (Matthew G. Crowson) is studying the mechanism of vascular endothelial growth factor (VEGF) promoter regulation following hypoxia by RTEF-1, a member of the TEA domain family of transcription factors. Specifically Matt has investigated Vestigial-like 4 (VGLL4), a cardiac-enriched transcription cofactor, and its interaction with the TEAD factors, including TEF-1 and RTEF-1 along with colleagues from the University of Ottawa Heart Institute. In previous studies, VGLL4 was used as bait to screen a mouse cardiac cDNA library in a yeast two-hybrid screen. The two-hybrid screen identified Interferon responsive factor 2 (IRF2) and interferon responsive factor 2 binding protein 2 (IRF2BP2) as a specific cofactors of VGLL4. Whereas IRF2 mRNA was not detected in normal tissues, Northern blot analysis showed that IRF2BP2 mRNA is enriched in cardiac and skeletal muscles. The IRF2BP2 and VGLL4 interaction was confirmed and further characterized by a mammalian two-hybrid assay in the myogenic C2C12 cell line. In addition, specific two conserved TEF-1-interacting TDU motifs in VGLL4 were identified as necessary for IRF2BP2/VGLL4 interaction since deletion of both motifs abolished and removal of one or the other motif reduced the protein-protein interaction.

Single Tube Luminometer Ravi Prakash Sahu, PhD
Postdoctoral Research Associate
Department of Pharmaceutical Sciences
Texas Tech University Health sciences Center,
School of Pharmacy, Amarillo Texas 79106
www.ttuhsc.edu/sop/pharmsci

The primary goal of my research is to delineate the molecular mechanisms for cancer prevention by naturally occurring compounds. I was granted a TD 20/20n luminometer to help us understand the transcriptional regulation of various transcription factors such as STAT-3 & NF-kB modulated by anticancer agents by luciferase assays. I will also be measuring the effects of SiRNAs on gene expression in cancer cells. In Summary, this instrument will help us to understand the mechanism of intracellular signaling and its regulation.

Single Tube Luminometer
Mohammed Balogun, Lecturer
Kampala International University
Biochemistry Department
Uganda
www.kampalaintuniv.net
This PhD research aims to explore and possibly find the cause(s) of observed regional diversity in HIV/AIDS immunomodulation. I hope to oligoGEArray assay to determine the level of expression of crucial immune response genes. This equipment will really help me in my work. Our laboratory is virtually empty but we have quite a number good research ideas which will truly help our people--Africans. Our University is new and is trying hard to train those who will change the continent's fortunes.

Single Tube Luminometer
Mihaela Gheorghiu, PhD
International Centre of Biodynamics
(Bio)Sensor Development
Bucharest, Romania
www.biodyn.ro
Investigation of luminescent/fluorescent reporter proteins (in engineered E. Coli). Investigate the ATP release from cell suspensions / monolayers. Confirmation of bacterial count performed within our centre using impedance spectroscopy and SPR assays. The assays I am running are Promega's Dual-Luciferase Assay, Luciferase Assay, Promega's ENLITEN ATP Assay, and Promega's Steady-Glo Assay for ATP Measurements, DNA Quantitation, bacterial count, chemi/fluorescent reporters. Intended on food and environmental quality control.

Single Tube Luminometer
Maria Montero, PhD
New York Blood Center
Blood Borne Parasitology Department
New York, New York
www.nybloodcenter.org
Plasmodium falciparum and Babesia divergens molecular biology
Check transitory and stable transformation with a plasmid that contains a luminiscence gen.
Using for check viability and surivence of transformed parasites.

 Single Tube Luminometer
Masahiro Sakagami
Assistant Professor
Virginia Commonwealth University
Pharamceutics, School of Pharmacy
Richmond, Virginia
http://www.pubinfo.vcu.edu/pharmacy/Faculty/FacDetail.asp?ID=85
The goals of my research program are to facilitate our understandings of lung biopharmaceutics and pharmacology for inhaled therapeutics and those of lung (patho)physiology under various diseases. Specifically, the current research project requiring the use of the 20/20n luminometer concerns pivotal biopharmaceutics issues of various inhaled corticosteroids (ICSs) treating asthmatic lungs. It has been shown that therapeutic efficacy of ICSs is not solely defined by their intrinsic anti-inflammatory potencies, such as glucocorticosteroid receptor binding affinity. It is hypothesized that local lung kinetics of aerosol deposition, dissolution and cellular uptake is also involved. Nevertheless, for their in vitro anti-inflammatory action assessments, conventional methods have employed non-confluent lung epithelial cells that are transiently transfected with inflammation-related genes (e.g., NFkB or AP1), which disregard such local lung cellular kinetics of ICS disposition. In this sense, we have recently developed a transfection protocol that enables the maintenance of sufficiently high levels of inflammation-related genes, even after the formation of tightly-packed, confluent lung epithelial cell (Calu-3) monolayers. Notably, these monolayers are capable of being grown under the air-interface culture, a culture fed only by basolateral medium, so that their semi-dry apical (mucosal) surfaces would allow direct aerosol deposition of ICSs into a limited volume of the cell lining fluid. This will give us a unique opportunity to assess trans-repression kinetics (e.g., onsets and potencies) of various ICSs upon their aerosol deposition and dissolution on the Calu-3 monolayer surfaces, using a reporter gene assay based on luciferase activity. Successful completion of this project will facilitate our understandings of ICS pharmacology and kinetics in lung. It is also believed that this unique and novel in vitro system is capable of leading to the discovery of novel therapeutic entities that exhibit anti-inflammatory actions for the treatment of a variety of other lung diseases via inhalation, by testing different classes of entities. It should be noted that this system is also capable of evaluating inflammatory responses and barrier function alterations of lung epithelium in response to inhalation aerosol deposition of more general substances, such as cigarette smokes and environment pollutants.
List of Assays = Luciferase activity from transient transfection of a reporter plasmid construct of inflammation-related transcription factor, pNFkB-Luc or pAP1-Luc, will be assayed with the cell lysates prepared from the Calu-3 monolayers following aerosol deposition and subsequent air-interface culture of various ICSs, such as fluticasone propionate, mometasone furoate, flunisolide, budesonide, triamcinolone acetonide and beclomethasone dipropionate. Preliminary transfection results obtained using a 20/20n luminometer owned by the School of Medicine at our University successfully induced the luciferase activity to 7.73±0.96 x 104 RLU (n=10) maintained in the tightly-packed confluent Calu-3 monolayers. Most importantly, one of the least soluble ICSs, fluticasone propionate, at 0.4 mM has shown to suppress such an induced activity following 24 hr incubation under the air-interface culture. All of these results should be quite convincing for a likely success of this project, given an award of own 20/20n luminometer in our laboratory for non-interrupted access for efficient progress and productivity in this research.
Description of Application = In this project, Calu-3 will be grown under the air-interface culture in the Transwell and transiently transfected with pNFkB-Luc or pAP1-Luc on Day 8 with an assist by a chelating agent, 2.5 mM ethyleneglycotetraacetic acid (EGTA). This protocol has been shown by us that efficient gene construct uptake and maintenance of high level transfection were feasible even after the formation of tightly-packed, confluent Calu-3 monolayers. On Day 12, the transfected Calu-3 monolayers will be subjected to aerosol deposition of various ICSs on their mucosal surface at different doses and sizes using the Andersen cascade impactor (ACI), a pharmacopeial testing instrument for particle size measurement of inhaler products. Briefly, each of the ICSs will be aerosolized from commercial inhalers and introduced into ACI, in which the semi-dry, confluent and transfected Calu-3 monolayers in the Transwell inserts will deposit the ICS aerosols by impaction. This unique system has been shown to enable accurate <0.2 mg of ICS deposition onto the 4.5 cm2 cell surfaces. Following continuing air-interface culture for various periods, the cell lysates will be prepared, according to the protocol given by Promega (Madison, WI), and their luciferase activity will be determined by an awarded 20/20n luminometer. This will enable characterization of trans-repression onsets and potencies for each of the ICSs at each dose and aerosol size deposition. Such trans-repression kinetics should be quite different from those reported from conventional in vitro methods using non-confluent cells and their ICS exposure under fully submerged incubation, by virtue of formidable barrier formation restricting ICS uptake into the cells and aerosol deposition and dissolution kinetics on the semi-dry Calu-3 surfaces with a limited volume of the cell lining fluid taken into account in the proposed studies. Obviously, our assessment with aerosol application should be a closer representation of real ICS actions in the organ following inhalation.

Single Tube Luminometer
Justin Hanes, Associate Professor
Johns Hopkins University
Chemical & Biomolecular Engineering Department
Baltimore, Maryland
http://www.jhu.edu/~cheme/hanes/
The primary goal of our lab is to design synthetic, non-viral gene carriers that can overcome various biological barriers and efficiently deliver therapeutic genes.  Gene therapy represents a promising strategy to combat diseases ranging from single gene defects to cancers, and many gene targets have been established. However, the development of safe and effective delivery strategies is widely recognized as the Achilles’ heel of gene therapy, due to the numerous extracellular and intracellular barriers. Our group is unique in our strategy to combine high resolution video microscopy and cellular biology to elucidate the dynamic behavior of gene carriers in complex biological environments.  The improved understanding can in turn facilitate the rational development of new generations of gene carriers. This approach is recently validated by the 1000-fold improvement in gene carrier transport across the highly viscoelastic mucus barrier, as well as the potential exploitation of a novel endocytic pathway and intracellular trafficking that minimizes gene degradation in cells. Our work in this area has been featured in over 30 scientific journals/news, including Nature Biotechnology and Journal of American Medical Association (JAMA) We are actively designing new generations of gene carriers for several formidable diseases, including cystic fibrosis (CF) as well as malignant cancers of the lung, ovary and liver.
List of Assays = (i) In vitro / in vivo transfection assay, (ii) siRNA knockdown study, (iii) promoter activity assay
The luminometer will be used as a broad platform to evaluate the gene transfer efficiency of our new generations of gene carrier. gWiz luciferase plasmid (Aldevron) will be condensed into small nanoparticles with different surface and biologically-active functionalities.  The efficacy of the gene carriers will be evaluated in vitro and in vivo using the Dual Luciferase Reporter Assay (Promega). One example is to study the efficiency of gene carriers administered to mucus covered lung epithelial cells for cystic fibrosis, where the thick mucus layer is a formidable barrier for gene therapy. The luciferase assay represents an important complementary tool to our current green fluorescence protein (GFP) based reporter protein, because it allows us to standardize our results to the luciferase assay commonly used in the field, and facilitate much higher throughput of in vivo testing due to auto-fluorescence in animals. The ease of the luciferase assay will greatly aid our development of new generations of efficient gene carriers. As an alternative to gene therapy, we are also researching on the development of therapies in cancer cells based on RNA-silencing (siRNA) technology. In collaboration with the Department of Gynecology and School of Public Health, we develop new polymeric carriers that can achieve targeted delivery of siRNA to ovarian and lung cancer cells. The luminometer allows us to rapidly measure the efficiency of siRNA delivery by measuring the luciferase knockdown in cells and mice stably transfected with a constitutively expressing luciferase vector. Another goal in our laboratory is to study the effects of specific promoters on expression profiles of therapeutic genes.  For example, it is known that certain promoters have higher activity in specific types of cancers.  This affords further improvement in the expression of genes in targeted cells.  The luciferase assay represents a high-throughput platform for us to scan for optimal promoters in a variety of cell lines.

Single Tube Luminometer
Melissa Rowland-Goldsmith, Assistant Professor
Chapman University
Biological Sciences Department
Orange, California
I am an assistant tenure track biology professor at a small liberal arts university where I am the only molecular biologist.   My university is very interested in starting a biochemistry major in which I will actively participate.  I have recently been teaching the senior capstone molecular genetics research class.  The students in that class have worked on research projects directly related to my research interests. My research focuses on the tumor promoting role of the growth factor, Transforming Growth Factor-Beta (TGF-Beta, on pancreatic cancer, which is still the 4th leading cause of cancer related deaths in the USA.  Previous research has shown that human pancreatic cancer cells over-express both TGF-beta and Vascular Endothelial Growth Factor (VEGF).  The over-expression of both of these proteins is correlated with decreased patient survival.  A main reason that pancreatic cancer is so aggressive is because the tumors are vascularized.  The growth and spread of cancer cells is dependent on angiogenesis.  VEGF is known to be the most potent angiogenic factor in promoting angiogenesis in pancreatic cancer.  My laboratory has recently shown that TGF-beta increases VEGF expression in pancreatic cancer cells. At this point, the molecular mechanism responsible for the induction of VEGF in pancreatic cancer is not known.  The purpose of this research project is to investigate the molecular mechanism promoting angiogenesis in pancreatic cancer by studying the up-regulation of VEGF gene transcription by TGF-beta.  We will determine the minimal VEGF promoter sequence that is TGF-beta responsive in pancreatic cancer cells treated with or without TGF-beta by generating several luciferase reporter constructs that contain sequential deleted promoter DNA sequences.  We will then transfect these constructs in pancreatic cancer cells treated with or without TGF-beta to determine the region of the promoter containing a potential Smad binding element that is necessary to increase VEGF transcription via the TGF-beta induced pathway.
List of Assays = dual reporter luciferase assay
We will study the VEGF promoter deletion constructs in response with or without TGF-beta treatment in  pancreatic cancer cell lines.  Specifically, we will do transient transfection assays using both the 5' deletion pGL3 luciferase reporter construct as well as an internal control which is the renilla luciferase vector so we can normalize the data.  The positive control will be cells transfected with the internal renilla luciferase vector and pGL3 control vector which contains both a strong promoter and enhancer to drive the expression of the luciferase gene.  The negative control will be cells transfected with the renilla luciferase control and pGL3 basic vector that does not contain any promoter or enhancer DNA sequence.  The transfected cells will be treated with or without TGF-beta ligand for 12-48 hours.  The cells will be lysed and we will use Promega reagents to do dual firefly luciferase and renilla luciferase assays using this 20/20 n luminometer.

 Single Tube Luminometer
William Baldwin, Associate Professor
Clemson University
Clemson Institute of Environmental Toxicology
Pendleton, South Carolina
http://www.clemson.edu/entox/index.htm
It is estimated that 106,000 people die each year from adverse drug reactions.  Many of these drug reactions are caused by concomitant exposure to other drugs, alternative medicines, pesticides, contraindicated foods.  Our goal is to identify and determine the potential for a number of xenobiotics such as pharmaceuticals, pesticides, nutraceuticals, and alternative medicines to cause drug-drug or toxicant-drug interactions by inducing P450 enzymes.  Identification of the chemicals that induce P450s will help us predict adverse drug interactions and save lives.
List of Assays = Dual-Glo luciferase, Steady Glo luciferase, P450-Glo CYP3A4, 1A1, 1A2, 2C8, 2C9, 2C19, and to a lesser extent 3A7 and 2D6.
We investigate the potential for drug-drug interactions by determining the (1) toxicants and alternative medicines that activate the constitutive androstane receptor (CAR) or the pregnane X-receptor (PXR), (2) determining the P450s induced in cell systems and in wild-type (CAR/PXR +/+), CAR-null, PXR-null, and mice humanized for CAR and PXR, and (3) determining whether there are sexually dimorphic differences in P450 induction. CAR and PXR are xenosensing nuclear receptors that recognize high concentrations of foreign chemicals and some endobiotics in our bodies and in turn induce drug metabolizing enzymes such as the P450s.  We will perform in vitro screening assays (about 48 luciferase assays per week) to determine chemicals that may activate these nuclear receptors, and then follow-up with dose-response curves.  Chemicals that activate at concentrations of concern will then be provided to colonies of wild-type and nuclear receptor null mice to determine if these chemicals have the potential to activate CAR or PXR in vivo.  Further studies will be performed in humanized mice or human primary cells to determine the relevance of our data to human health.  Real-time quantitative PCR, Western blots, and P450-Glo assays will be used to determine the P450s induced, or in some cases the P450s inhibited by the toxicant of interest.

 Single Tube Luminometer
Xiongbin Lu, Assistant Professor
University of South Carolina
Biologial Sciences Department
Columbia, South Carolina
In my laboratory, we are interested in the p53 tumor suppressor and its signaling pathways. p53 activity is tightly controlled in the cell. To elucidate how p53 is regulated, we will measure its activity by luciferase assays. We will transfect cells with luciferase expression vectors driven by the promoters of p53-targeting genes such as p21, Mdm2 and Bax promoters. P53 transcriptional activity will be determined by measuring luciferase activity in each cell sample. Turner 20/20n luminometer will greatly facilitate our research and provides a precise way for us to clarify the mechanisms by which p53 is activated or suppressed.

Single Tube Luminometer
Dr. Robert L. Caldwell is an Assistant Professor of Orthopaedics and Rehabilitation, and Cancer Biology at Vanderbilt University School of Medicine. He was granted a TD 20/20n luminometer to help understand the transcriptional regulation of the bone morphogenic protein receptor-2 (BMPR2) in osteosarcomas and bone metabolic diseases. Elucidation of mechanisms underlying BMPR2 gene expression will deepen our understanding of osteosarcoma signal transduction pathways as well as molecular events giving rise to osteoporosis and osteoarthritis.

 Single Tube Luminometer
Dr. Dennis De Luca is an associate professor at Ohio Northern University. The Department of Biological and Allied Health Sciences has 18 full-time professors with nearly 300 majors. The students major in Clinical Laboratory Sciences, environmental studies, molecular biology, forensic biology and as preprofessional biology majors. The basic science departments serve the needs of the largest pharmacy program (Pharm.D.)in the state of Ohio. The courses in molecular, cell, microbiology, toxicology, genetics will be enhanced using assays for DNA quantitation, reporter gene systems,and other luminescence assays with the use of this luminometer.

Single Tube Luminometer
Dr. Geraldine Misquith is the Biology Department's Laboratory Coordinator at West Chester University in West Chester, PA. She was granted a 20/20ⁿ luminometer to help their undergraduate and graduate students perform chemiluminescent reporter gene assays for the combined detection of luciferase and ß-galactosidase. One application includes the evaluation of the functional promoter under basal and hypertonic conditions in mouse lens epithelial cells.

 Single Tube Luminometer
Dr. William Kim is an Assistant Professor at Columbia University Medical Center in New York, NY. He was granted a 20/20ⁿ luminometer that will help to measure subcellular localization of luminescent potential target marker construct molecules in transfected cells. Their research is focused on studying the molecular and cellular mechanisms of diabetes and cardiovascular diseases.

Single Tube Luminometer
Dr. Lyudmila Dubovskaya is a scientist at the Institute of Biophysics and Cell Engineering at the National Academy of Sciences in Minsk, Belarus. Dr. Dubovskaya was granted a TD-2020 luminometer that will help further their research focused on molecular and membrane mechanisms of photobiological reactions, receptor interactions and intracellular signaling in plant and animal systems. They are currently investigating the role of intracellular calcium concentration changes in regulation of plant metabolism under stress conditions.

Single Tube Luminometer
Dr. Natalie Kuldell teaches in the Biological Engineering Department at MIT. She was granted a TD-20/20 Luminometer to use in her undergraduate Laboratory classes that introduce students to expression engineering using RNAi and Microarray technologies. The primary applications will be measuring the effect of siRNAs on gene expression in Hela cells.

Single Tube Luminometer
Dr. Rafiq Islam is at Northwest Missouri State University in the Department of Chemistry and Physics. He was granted a TD-20/20 Luminometer to help understand how various factors regulate transcription of the polycystic kidney disease-1 (PKD1) gene. Mutations in the human PKD1 gene are responsible for the vast majority of autosomal dominant polycystic kidney disease (ADPKD) cases. He will be looking at factors that modulate transcription of the PKS1 promoter.

Single Tube Luminometer
Dr. Margaret Rice is an Associate Professor at the NYU School of Medicine in the Department of Physiology and Neuroscience. She was granted a TD-20/20 Luminometer for her laboratory work to determine how H202 activates K-ATP channels. Understanding the mechanism by which H202 modulates neuronal activity and transmitter release will provide new information about brain neurochemistry and neurophysiology. The findings will shed light on the underlying causes of neurodegenerative disorders like Parkinson's disease. View Abstract

Single Tube Luminometer
Dr. Stephen St. Jeor at the University of Nevada in Reno was granted a TD-20/20 Luminometer. His laboratory is part of the Microbiology Department at the School of Medicine. He is studying the pathogenesis of virus diseases and his lab is researching the area of virus pathogenesis. They are interested in the regulation of human cytomegalovirus (HCMV) latency, the pathogenesis of New World Hantaviruses, and characterization of strains of Crimean Congo hemorrhagic fever (CCHF) from Africa. HCMV is a member of the herpes group of viruses, and infects a high percentage of the human population.

Single Tube Luminometer
Professor Gary Linidquester, Chairman of the Biology Department and Rhodes College, has been granted a TD-20/20 Luminometer for use in three research laboratories. One lab is trying to understand the role of interluekin-10 gene of Epstein-Barr virus, another lab studies cell-cycle regulation in yeast and the third lab studies wall morphogenesis and development in filamentous fungi. The TD-20/20 will also be used in Molecular Biology courses.

Single Tube Microplate Luminometer
Dr. Levi Downs, Jr. is an Assistant Professor in the Dept of Obstetrics, Gynecology and Women's Health at the University of Minnesota Medical School. Dr. Downs was granted a GloRunner that will help to evaluate the in-vivo use of siRNA targeting the human papilloma viral proteins E6 and E7 for the treatment of cervical cancer. The human papilloma virus (HPV) proteins E6 and E7 are necessary for the initiation and maintenance of the carcinogenic phenotype of cervical cancer. The GloRunner will be used for Genospectra™ branched DNA assays which will be used to evaluate the gene knockdown by siRNA. Gynecological oncology fellows will also use the luminometer as part of their research training.

Single Tube Microplate Luminometer
Professor Carlos Stocco of the Yale University OBGYN Department was granted a Reporter Microplate Luminometer for working with Luciferase Assays. His goal is to characterize the effect of PGF2&#61537 on cyp19 expression and to investigate in vivo transcription factors which may regulate basal expression of P450arom and to identify those that may mediate the effect of PGF2a on cyp19 transcription.

Single Tube Microplate Luminometer
Dr. Barry Hudson of Columbia University Department of Surgery was granted a Reporter Luminometer. Dr. Hudson is working to identify the role of the RAGE gene in the pathogenic processes of inflammatory based diseases including cardiovacular disease and cancer. Identifying how the gene works from the transcriptional level through to the protein level is essential for designing therapeutics.

Single Tube Microplate Luminometer
Professor Mariana Foldavari of the University of Saskatchewan was granted a Reporter Microplate Luminometer for use at the College of Pharmacy and Nutrition. Keratinocytes transfected with luciferase coding plasmid will be evaluated for luciferase expression using the Reporter. Adequate levels of luciferase expression will be reflective of the potential for delivering therapeutic genes into the skin or transdermally. The Reporter will be used in the research and education of students at the University.

Single Tube Microplate Luminometer
Professor Katia Gysling at the Catholic University of Chile in Santiago was granted a Model 20e Luminometer. She is in the Department of Cell and Moleular Biology and is studying the molecular and functional basis of limbic system activity. The research centers on the role of peptides corticotropin releasing hormone and urocortin in anxiety and drug additicion. The Luminometer will be used to measure luciferase activity in the study of the regulation of UCN gene transcription.

Single Tube Luminometer
Dr. Leticia Carniero at the Universidade do Estado do Rio de Janeiro has been granted a tube Luminometer. Her research is conducted in the department of Microbiology and Immunology. They will be using this instrument to study proteins in the context of infections by intestinal (Shigella and Salmonella) and respiratory (pseudomonas) pathogens. This is the first luminometer at this University and other departments are interested in using it.