Guideline on Genetically Modified Revised Final
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Rnai in Primary Cells and Difficult-To-Transfect Cell Lines
Automated High Throughput Nucleofection® RNAi in Primary Cells and Difficult-to-Transfect Cell Lines Claudia Merz, Bayer Schering Pharma AG, Berlin, Germany; Andreas Schroers, amaxa AG, Cologne, Germany; Eric Willimann, Tecan AG, Männedorf, Switzerland. Introduction Materials & Methods - Workflow Using primary cells for RNAi based applications such as target identification or – validation, requires a highly efficient transfection displaying the essential steps of the automated Nucleofector® Process: technology in combination with a reliable and robust automation system. To accomplish these requirements we integrated the amaxa 1. Transfer of the cells to the Nucleocuvette™ plate, 96-well Shuttle® in a Tecan Freedom EVO® cell transfection workstation which is based on Tecan’s Freedom EVO® liquid handling 2. Addition of the siRNA, (Steps 1 and 2 could be exchanged), platform and include all the necessary components and features for unattended cell transfection. 3. Nucleofection® process, 4. Addition of medium, Count Cells 5. Transfer of transfected cells to cell culture plate for incubation ® Nucleofector Technology prior to analysis. Remove Medium The 96-well Shuttle® combines high-throughput compatibility with the Nucleofector® Technology, which is a non-viral transfection method ideally suited for primary cells and hard-to-transfect cell lines based on a combination of buffers and electrical parameters. Nucleocuvette Plate Add Nucleofector +– The basic principle and benefits of the (empty) Solution Cell of interest Gene of interest Nucleofector® -
Widespread Gene Transfection Into the Central Nervous System of Primates
Gene Therapy (2000) 7, 759–763 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Widespread gene transfection into the central nervous system of primates Y Hagihara1, Y Saitoh1, Y Kaneda2, E Kohmura1 and T Yoshimine1 1Department of Neurosurgery and 2Division of Gene Therapy Science, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan We attempted in vivo gene transfection into the central ner- The lacZ gene was highly expressed in the medial temporal vous system (CNS) of non-human primates using the hem- lobe, brainstem, Purkinje cells of cerebellar vermis and agglutinating virus of Japan (HVJ)-AVE liposome, a newly upper cervical cord (29.0 to 59.4% of neurons). Intrastriatal constructed anionic type liposome with a lipid composition injection of an HVJ-AVE liposome–lacZ complex made a similar to that of HIV envelopes and coated by the fusogenic focal transfection around the injection sites up to 15 mm. We envelope proteins of inactivated HVJ. HVJ-AVE liposomes conclude that the infusion of HVJ-AVE liposomes into the containing the lacZ gene were applied intrathecally through cerebrospinal fluid (CSF) space is applicable for widespread the cisterna magna of Japanese macaques. Widespread gene delivery into the CNS of large animals. Gene Therapy transgene expression was observed mainly in the neurons. (2000) 7, 759–763. Keywords: central nervous system; primates; gene therapy; HVJ liposome Introduction ever, its efficiency has not been satisfactory in vivo. Recently HVJ-AVE liposome, a new anionic-type lipo- Despite the promising results in experimental animals, some with the envelope that mimics the human immuno- gene therapy has, so far, not been successful in clinical deficiency virus (HIV), has been developed.13 Based upon 1 situations. -
Introduction to Flow Cytometry Principles Data Analysis Protocols Troubleshooting
Flow Cytometry ipl.qxd 11/12/06 11:14 Page i Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Your first choice for antibodies! Flow Cytometry ipl.qxd 11/12/06 11:14 Page ii Flow Cytometry ipl.qxd 11/12/06 11:14 Page iii Introduction to Flow Cytometry Principles Data analysis Protocols Troubleshooting By Misha Rahman, Ph.D. Technical advisors Andy Lane, Ph.D. Angie Swindell, M.Sc. Sarah Bartram, B.Sc. Flow Cytometry ipl.qxd 11/12/06 11:14 Page 2 Preface How can I explain what flow cytometry is to someone that knows nothing about it? Well, imagine it to be a lot like visiting a supermarket. You choose the goods you want and take them to the cashier. Usually you have to pile them onto a conveyor. The clerk picks up one item at a time and interrogates it with a laser to read the barcode. Once identified and if sense prevails, similar goods are collected together e.g. fruit and vegetables go into one shopping bag and household goods into another. Now picture in your mind the whole process automated; replace shopping with biological cells; and substitute the barcode with cellular markers – welcome to the world of flow cytometry and cell sorting! We aim to give you a basic overview of all the important facets of flow cytometry without delving too deeply into the complex mathematics and physics behind it all. For that there are other books (some recommended at the back). -
Immunology and Flow Cytometry Lab
Client/Submitter Bill to Client/Submitter Bill to Patient Insurance (see requirements below) Children's Hospital Colorado Specimen Shipping Address: Department of Pathology & Laboratory Medicine Children's Hospital Colorado Flow Cytometry & Immunology Lab Requisition Clinical Laboratory - Room B0200 Phone (720) 777-6711 13123 E. 16th Ave Fax (720) 777-7118 Aurora, CO 80045 FAILURE TO COMPLETE BELOW FIELDS WILL DELAY RESULTS ***PLEASE PROVIDE COMPLETE BILLING INFORMATION** Contact Information Submitting Institution Name (Submitter) Submitting Institution Address Street City, State, Zip Phone Result Fax Client Specimen Label (if available) Internal Specimen Label Patient Information Last Name First Name Middle I Birthdate (MM/DD/YYYY) Sex Ordering Provider (Last, First, and Middle Initial) Ordering Provider Phone Ordering Provider NPI Specimen Information Date Collected (MM/DD/YY) Client External ID ICD-10 Code(s) □ Blood 1 □ Bone Marrow Time Collected (HHMM) Draw Type 2 □ Tissue-Fresh: AM / PM 3 □ Body Fluid FAILURE TO COMPLETE WILL DELAY RESULTS Bill To: □ Billing Facility and Address same as Submitter Listed Billing Contact Information: Billing Facility and Address are DIFFERENT than Submitter Listed, Bill To: Name: ___________________________________________________________ Institution Name: _______________________________________________________ Email: ___________________________________________________________ Address (incl City, State, Zip): ______________________________________________ Phone: ___________________________________________________________ -
Overview of Flow Cytometry and Its Uses for Cell Analysis and Sorting
Principles of flow cytometry: overview of flow cytometry and its uses for cell analysis and sorting Shoreline Community College BIOL 288 Flow Cytometry • What is Flow Cytometry? – Measurement of cells or particles in a fluid stream • The first commercial cytometer was developed in 1956 (Coulter counter) • The first fluorescent based cytometer was developed in 1968. • In 1978 the term Flow Cytometry was adopted and companies began to manufacture commercial instrument systems Flow Cytometry • Flow cytometry uses fluorescent light and non-fluorescent light to categorize and quantify cells or particles. • An Instrument collects and measures multiple characteristics of individual cells within a population as they pass through a focused beam of light. • Modern flow cytometers are powerful tools; at rates of several thousand to 10’s of thousand cells per second, quantifiable data on complex mixtures can be obtained revealing the heterogeneity of a sample and its many subsets of cells. • Examples of use Immunophenotyping | Cell proliferation | Tracking proteins or genes Activation studies |Immune response | Apoptosis Cell sorting All of the above and in addition, single to multiple populations are physically separated and purified from a mixed sample for downstream assays Flow Cytometry Where is flow cytometry used? Biomedical research labs Immunology, Cancer Biology, Neurobiology, Molecular biology, Microbiology, Parasitology Diagnostic Laboratories Virology - HIV/AIDS, Hematology, Transplant and Tumor Immunology, Prenatal Diagnosis Medical Engineering Protein Engineering, Microvessicle and Nanoparticles Marine and Plant Biology Fluorescence • Fluorescent molecules emit light energy within a spectral range • Fluorescent Dyes and Proteins are selective and highly sensitive detection molecules used as markers to classify cellular properties. • Monoclonal antibodies and tetramers when coupled with fluorescent dyes can be detected using flow cytometry. -
Flow Cytometric Determination for Dengue Virus-Infected Cells: Its Application for Antibody-Dependent Enhancement Study
Flow Cytometric Determination for Dengue Virus-Infected Cells: Its Application for Antibody-Dependent Enhancement Study Kao-Jean Huang*, Yu-Ching Yang*, Yee-Shin Lin*, Hsiao-Sheng Liu*, Trai-Ming Yeh**, Shun-Hua Chen*, Ching-Chuan Liu*** and Huan-Yao Lei*! *Departments of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan **Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan ***Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan Abstract The theory of antibody-dependent enhancement plays an important role in the dengue virus infection. However, its molecular mechanism is not clearly studied partially due to lack of a sensitive assay to determine the dengue virus-infected cells. We developed a flow cytometric assay with anti-dengue antibody intracellular staining on dengue virus-infected cells. Both anti-E and anti-prM Abs could enhance the dengue virus infection. The anti-prM Ab not only enhanced the dengue virus infected cell mass, but also increased the dengue virus protein synthesis within the cells. The effect of anti-prM Ab- mediated enhancement on dengue virus infection is serotype-independent. We concluded that the target cell-based flow cytometry with anti-dengue antibody intracellular staining on dengue virus- infected cells is a sensitive assay to detect the dengue virus infected cells and to evaluate the effect of enhancing antibody on dengue virus infection on cell lines or human primary monocytes. Keywords: Dengue, enhancing antibody, ADE, flow cytometry. Introduction characterized by abnormalities of haemostasis and increased vascular permeability, which in Dengue is an acute infectious disease caused some instances results in DSS. -
Food and Drug Law Journal
FOOD AND DRUG LAW JOURNAL EDITOR IN CHIEF Judy Rein EDITORIAL ADVISORY BOARD CHAIR VICE CHAIR FACULTY ADVISOR Laurie Lenkel Robert Giddings Joseph A. Page FDA – OC Hutchison PLLC Georgetown University Law Center ________________________________ Anthony Anscombe James Flaherty Francis Palumbo Sedgwick LLP Fresenius Medical University of Maryland School of Pharmacy Peter Barton Hutt Abraham Gitterman Covington & Burling Arnold & Porter LLP Sandra Retzky FDA – CTP Barbara Binzak Kimberly Gold Blumenfeld Norton Rose Fulbright Joan Rothenberg Buchanan Ingersoll & LLP FDA - CFSAN Rooney PC John Johnson Jodi Schipper Catherine Clements FDA Imports FDA – CDER Express Scripts Alan Katz Christopher van Gundy Kellie Combs toXcel, LLC Keller and Heckman Ropes & Gray LLP Sara Koblitz James Woodlee Nathan Cortez Fish & Richardson Kleinfeld Kaplan & Becker LLP Southern Methodist University Valerie Madamba Emily Wright Blue Apron Pfizer Brian Dahl Dahl Compliance Alan Minsk Kimberly Yocum Consulting LLC Arnall Golden Gregory TC Heartland LLC LLP Sandra dePaulis Lowell Zeta FDA – CVM Nicole Negowetti Hogan Lovells The Good Food Ian Fearon Institute Patricia Zettler British American Tobacco Georgia State James O’Reilly University Law School University of Cincinnati OFFICERS OF THE FOOD AND DRUG LAW INSTITUTE CHAIR: Allison M. Zieve, Public Citizen Litigation Group VICE CHAIR: Jeffrey N. Gibbs, Hyman, Phelps & McNamara, P.C. TREASURER: Frederick R. Ball, Duane Morris LLP GENERAL COUNSEL/SECRETARY: Joy J. Liu, Vertex Pharmaceuticals IMMEDIATE PAST CHAIR: Sheila Hemeon-Heyer, Heyer Regulatory Solutions LLC PRESIDENT & CEO: Amy Comstock Rick GEORGETOWN UNIVERSITY LAW CENTER STUDENT EDITOR IN CHIEF Dana Shaker STUDENT MANAGING EDITORS Jacob Klapholz Christine Rea STUDENT NOTES EDITOR SYMPOSIUM EDITOR Lauren Beegle Alexander P. -
The Art of Transfection (Poster / Pdf)
TRANSDUCTION NON-VIRAL TRANSFECTION Transduction is the process of using vectors including retroviruses, lentiviruses, adenoviruses, PACKAGE DELIVERY: Chemical Chemical transfection adeno-associated viruses, or hybrids to deliver genetic payloads into cells. Generally, a plasmid transfection reagent containing mRNA carrying genes flanked by viral sequences is first transfected into a producer cell with other reagent virus-associated (packaging) plasmids. In the producer cells, virions form that contain the gene The Art of Transfection of interest. For safety, no plasmid used in the process contains all of the necessary sequences Inserting genetic material into mammalian and insect cells without killing them can be a challenge, CHEMICAL TRANSFECTION for virion formation, and only the plasmid carrying the gene of interest contains signals that but scientists have developed several ways to perform this intricate task. Transfection is the process of Functional proteins or allow it to be packaged into virions. Researchers then extract, purify, and use the virions from Complexation structural components released Chemical carriers represent the most straightforward and widespread tools for gene delivery the producer cells to insert DNA into other cells to stably or transiently express the DNA of introducing nucleic acids (plasmid DNA or messenger, short interfering, or micro RNA) into a cell. from cell or into cytoplasm experiments in mammalian cells. Chemical transfection experiments follow a simple workflow and interest. The transferred genetic material, which lacks viral genes, cannot generate new viruses. Researchers accomplish this with nonviral methods (chemical or physical transfection), or with viral provide high efficiency nucleic acid delivery for the most commonly used cells as well as many methods, commonly referred to as transduction. -
Flow Cytometry: Basic Principles and Applications
Critical Reviews in Biotechnology ISSN: 0738-8551 (Print) 1549-7801 (Online) Journal homepage: http://www.tandfonline.com/loi/ibty20 Flow cytometry: basic principles and applications Aysun Adan, Günel Alizada, Yağmur Kiraz, Yusuf Baran & Ayten Nalbant To cite this article: Aysun Adan, Günel Alizada, Yağmur Kiraz, Yusuf Baran & Ayten Nalbant (2016): Flow cytometry: basic principles and applications, Critical Reviews in Biotechnology, DOI: 10.3109/07388551.2015.1128876 To link to this article: http://dx.doi.org/10.3109/07388551.2015.1128876 Published online: 14 Jan 2016. Submit your article to this journal Article views: 364 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ibty20 Download by: [Universidad Autonoma Metropolitana] Date: 09 May 2016, At: 17:45 http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–14 ! 2016 Taylor & Francis. DOI: 10.3109/07388551.2015.1128876 REVIEW ARTICLE Flow cytometry: basic principles and applications Aysun Adan1*,Gu¨nel Alizada2*, Yag˘mur Kiraz1,2*, Yusuf Baran1,2, and Ayten Nalbant2 1Faculty of Life and Natural Sciences, Abdullah Gu¨l University, Kayseri, Turkey and 2Department of Molecular Biology and Genetics, I_zmir Institute of Technology, I_zmir, Turkey Abstract Keywords Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a Apoptosis, cytokines, flow cytometer, single cell such as size and granularity simultaneously as the cell flows in suspension through a fluorescence, fluorescent-activated cell measuring device. Its working depends on the light scattering features of the cells under sorting, histogram, immunophenotyping, investigation, which may be derived from dyes or monoclonal antibodies targeting either light scatter extracellular molecules located on the surface or intracellular molecules inside the cell. -
Micro-RNA Modulation of Insect Virus Replication Verna Monsanto-Hearne and Karyn N
Micro-RNA Modulation of Insect Virus Replication Verna Monsanto-Hearne and Karyn N. Johnson* School of Biological Sciences, University of Queensland, Brisbane, Australia. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.034.061 Abstract Saleh, 2012; Xu and Cherry, 2014; Mussabekova Te outcome of virus infection in insects is impacted et al., 2017). Many molecular components medi- by regulation of both host and virus gene expres- ate and are mediated by this host–virus cross-talk, sion. A class of small RNAs called micro-RNAs including microRNAs. (miRNA) have emerged as important regulators of microRNAs (miRNAs) are a large class of highly gene expression that can infuence the outcome of conserved, ≈ 22 nt non-coding RNAs that regulate virus infection. miRNA regulation occurs at a com- gene expression. Compared to the more upstream paratively late stage of gene expression, allowing for regulatory mechanisms such as transcriptional rapid control and fne-tuning of gene expression regulation and chromatin remodelling, regulation levels. Here we discuss the biogenesis of miRNAs by miRNA occurs at a later stage of gene expres- from both host and virus genomes, the interactions sion, allowing for rapid control and fne-tuning of that lead to regulation of gene expression, and the gene expression levels (Chen et al., 2013). Comple- miRNA–mRNA interactions that lead to either mentary binding of at least the seed region (second antivirus or provirus consequences in the course of to eighth nucleotide from the 5′-end) of the ≈ 22 nt virus -
SY3200 Cell Sorter True Innovation Inside
SY3200 Cell Sorter true innovation inside Europe True Innovation The SY3200™ system is one of the most innovative and versatile Flow Cytometry Research platforms available in the market. True innovation drives superior performance and versatility in the SY3200 system. From optics to electronics, revolutionary technology provides solutions that enable the complex applications of today and tomorrow. Advancements in discovery of new markers and new dyes have improved the ability to identify more populations in a single assay. This has advanced the use of flow cytometry in an increasing number of applications as well as challenged instrumentation to continue to adapt to new parameters. The SY3200 has been designed to easily adapt to the ever increasing need for flexibility and sensitivity in new applications. Whereas some systems are merely evolutionary in design, the SY3200 brings revolutionary, innovative and cutting edge technology to elevate the science of flow cytometry. The innovative and flexible laser delivery array allows for ultimate laser compatibility while the unique reflective collection optics ensure maximum fluoresence collection efficiency. Powerful, Sony designed electronics enable collection of high resolution data and ultra high speed cell sorting while maintaining superior sensitivity. Plug and Play PMTs in the Scalable PMT Array (SPA) module provide the ultimate flexibility in collection options while minimizing cost. The SY3200 provides true innovation to deliver a comprehensive cell sorting solution that meets the challenges of the increasing complexity of research flow cytometry applications. Ultimate Configurability The SY3200 platform is designed for configurability to adapt to the demanding needs of a variety of sorting labs. Unique to any system in flow cytometry, the SY3200 is expandable to a dual system layout, effectively enabling two systems in one. -
Genetic Engineering: Moral Aspects and Control of Practice
Journal of Assisted Reproduction and Genetics, VoL 14. No. 6, 1997 NEWS AND VIEWS REPRODUCTIVE HEALTH CARE POLICIES AROUND THE WORLD Genetic Engineering" Moral Aspects and Control of Practice INTRODUCTION outline the concept of gene therapy with regard to the ethical aspects involved. Since the time of Hippocrates, physicians have sought to understand the'mechanisms of disease development for the purposes of developing more HISTORY effective therapy. The application of molecular biol- ogy to human diseases has produced significant The following is a concise review of the history discoveries about some of these disease states. of gene therapy. Extensive reviews can be found Gene transfer involves the delivery to target cells elsewhere (1). The human species has for many of an expression cassette made up of one or more generations practiced genetic manipulation on genes and the sequence controlling their expres- other species. Examples can be seen in the breed- sion. The process is usually aided by a vector that ing of animals and plants for specific intent, such helps deliver the cassette to the intracellular site as, corn, roses, dogs, and horses. The term "genet- where it can function appropriately. Human gene ics" was first used in 1906, and the term "genetic therapy has moved from feasibility and safety stud-. engineering" originated in 1932. Genetic engi- ies to clinical application more rapidly than was neering was then defined as the application of expected. The development of recombinant DNA genetic principles to animal and plant breeding. technology brought science to the brink of curing The term "gene therapy" was adopted to distin- previously untreatable diseases in a relatively short guish itself from the ominous, germ-line perception period of time.