Bacterial Transformation
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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® -
Improving DNA Plasmid Production in Escherichia Coli
Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER (Under the Direction of Mark A. Eiteman) ABSTRACT The ability to produce large quantities of plasmid DNA is imperative for wide scale availability of DNA vaccines. Large scale, high yield production relies on the synergy between host strain, plasmid, medium and production scheme. Screening as many variables as quickly and cost effectively as possible is the goal. In this study, Escherichia coli strains were transformed with two plasmids and screened for plasmid yield in shake flasks in chemically defined medium supplemented with either glucose or glycerol. High yield candidates were grown in feed batch fermentations at two specific growth rates, µ = 0.14 h-1 and µ = 0.24 h-1. As predicted, high production in shake flasks was predictive of high production in fermentations. Using our media and process, we were able to reach volumetric yields of approximately 600 mg/L and specific yields of approximately 17.82 mg/g, regardless of growth rate. We were also able to increase productivity (mg/Lh) over 30%. INDEX WORDS: E. coli, fed-batch, gene therapy, plasmid production Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER B.S., Biological Engineering, University of Georgia, 1998 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2007 © 2007 Adam Singer All Rights Reserved Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER Major Professor: Mark A. Eiteman Committee: Elliot Altman Sidney Kushner Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2007 iv DEDICATION To my wife Dana and my daughter Sydney-Rose. -
Synthesis of a Contiguous 32-Kb Polyketide Synthase Gene Cluster
Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster Sarah J. Kodumal, Kedar G. Patel, Ralph Reid, Hugo G. Menzella, Mark Welch, and Daniel V. Santi* Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545 Communicated by Robert M. Stroud, University of California, San Francisco, CA, September 17, 2004 (received for review July 19, 2004) To exploit the huge potential of whole-genome sequence infor- Our efforts to this end stemmed from a desire to develop mation, the ability to efficiently synthesize long, accurate DNA heterologous expression of large polyketide synthase (PKS) sequences is becoming increasingly important. An approach pro- genes in Escherichia coli. Type I modular PKS genes encode the posed toward this end involves the synthesis of Ϸ5-kb segments of giant enzymes (among the largest proteins known) that synthe- DNA, followed by their assembly into longer sequences by con- size polyketide natural products such as erythromycin, epothi- ventional cloning methods [Smith, H. O., Hutchinson, C. A., III, lone, and tacrolimus (12). These genes reside within the high Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, GϩC genomes of the actinomycete and myxobacterial groups of 15440–15445]. The major current impediment to the success of this prokaryotes and encode proteins with multiple sets, or modules, tactic is the difficulty of building the Ϸ5-kb components accurately, of active sites (domains). Each module catalyzes the assembly of efficiently, and rapidly from short synthetic oligonucleotide build- a specific two-carbon-unit component of the polyketide product. ing blocks. -
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. -
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. -
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 -
A Visual Demonstration of Plasmid DNA Preparation from Bacteria
HOW-TO-'DO-IT A Visual Demonstration of Plasmid DNA Preparation from Bacteria * RALPH L. KEIL, LAURA K. PALMER Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 Unprecedented advances in unraveling mysteries of life have disease, or other areas the students or instructor want to develop. occurred as a result of the molecular biology revolution. Some Since many students know someone with diabetes (in many cases major achievements of this revolution include new methods for the they know a classmate who has diabetes or one of them may even production of therapeutic compounds, insights into mechanisms suffer from the disease), this example provides an opportunity to of gene function and regulation, and the complete sequencing of get students to actively participate in discussion, regardless of their the genomes of numerous organisms including humans. A critical academic abilities. foundation of this revolution is the ability to use the bacterium An alternative introduction is to discuss consequences of Escherichia coli (E. coli) as a factory to produce large amounts of sequencing the entire three billion base pairs of DNA of the virtually any DNA of interest by inserting it into a small, extrachro human genome (http://www.ornl.gov/sci/techresources/Human mosomal circle of DNA called a plasmid. The ease and reproducibil Genome/home.shtml). The current and potential uses of this ity of plasmid DNA isolation from bacteria permits numerous labs technology in forensic science, health care, and business decisions to conduct molecular biology experiments and greatly accelerates are all areas that may be discussed depending on what focus the progress in understanding complex biological processes. -
Control of Ph During Plasmid Preparation by Alkaline Lysis of Escherichia Coli
Analytical Biochemistry 378 (2008) 224–225 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Notes & Tips Control of pH during plasmid preparation by alkaline lysis of Escherichia coli Cheri Cloninger, Marilyn Felton 1, Bonnie Paul 1, Yasuko Hirakawa, Stan Metzenberg * Department of Biology, California State University, Northridge, Northridge, CA 91330, USA article info abstract Article history: Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ‘‘ghost bands” Received 9 March 2008 of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing Available online 11 April 2008 the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing. Ó 2008 Elsevier Inc. All rights reserved. The extraction of plasmids from Escherichia coli is one of the most 2. To each 1 ml of cell suspension, 1 ml of a lysis solution consist- commonly used methods in molecular biology, and detergent lysis of ing of 1% (w/v) sodium dodecyl sulfate (SDS) and 0.5 M L-argi- cells in 0.1 M sodium hydroxide (NaOH,2 final) is the most wide- nine (pH 11.7) is added, and the tube is capped and rocked spread approach [1,2]. The alkalinity denatures the chromosomal briefly to mix. The lysate is allowed to sit undisturbed for 5 min. -
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. -
Molecular Biology Services Price List Denmark; Valid from 01.09.2017
Molecular Biology Services Price List Denmark; valid from 01.09.2017 Gene Synthesis Service Delivery times for Gene Synthesis Service: Standard Genes up to 1000 bp: 6 business days; guaranteed: 12 business days Standard Genes ≤ 3000 bp: 15 - 20 business days Standard Genes > 3000 bp: 4 additional business days/kb; Subcloning: additional 5 - 10 business days Express Genes up to 1000 bp: 4 business days Express Genes up to 1500 bp: 6 business days; up to 2000 bp: 7 days; Express Genes up to 3000 bp: 11 business days; up to 4000 bp: 13 days; Express subcloning: 4 business days Delivery time for complex gene varies. Dependent on the complexity of the gene, usually it takes 5-10 business days longer than estimated for standard genes. Part ID Service Description Price [DKK] 5001-000010 Short Standard Genes (≤ 500 bp) 1,125.00 / gene 5001-000016 Standard Genes (501-1000 bp) 2.40 / base pair 5001-000012 Long Standard Genes (1001-3000bp) 2.40 / base pair 5001-000019 Long Standard Genes (3001-4000bp) 2.70 / base pair 5001-000119 Long Standard Gene (4001-5000bp) 3.00 / base pair 5001-000219 Long Standard Gene (5001-6000bp) 3.15 / base pair 5001-000319 Long Standard Gene (6001-7000bp) 3.53 / base pair 5001-000419 Long Standard Gene (7001-8000bp) 3.53 / base pair 5001-000519 Long Standard Gene (8001-9000bp) 3.53 / base pair 5001-000619 Long Standard Gene (9001-10000bp) 3.53 / base pair 5001-000719 Long Standard Gene > 10 kb on request 5001-000021 Complex Genes on request 5001-000230 Express Fee Short Standard Gene (≤ 500 bp) 3,000.00 / gene 5001-000232 -
Agrobacterium Tumefaciens-Mediated Genetic Transformation of the Ect-Endomycorrhizal Fungus Terfezia Boudieri
G C A T T A C G G C A T genes Technical Note Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri 1,2 1, 1 1, Lakkakula Satish , Madhu Kamle y , Guy Keren , Chandrashekhar D. Patil z , Galit Yehezkel 1, Ze’ev Barak 3, Varda Kagan-Zur 3, Ariel Kushmaro 2 and Yaron Sitrit 1,* 1 The Albert Katz International School for Desert Studies, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel; [email protected] (L.S.); [email protected] (M.K.); [email protected] (G.K.); [email protected] (C.D.P.); [email protected] (G.Y.) 2 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering and The Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel; [email protected] 3 Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel; [email protected] (Z.B.); [email protected] (V.K.-Z.) * Correspondence: [email protected]; Tel.: +972-8-6472705 Current address: Department of Forestry, North Eastern Regional Institute of Science & Technology, y Nirjuli, Arunachal Pradesh 791109, India. Current address: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, z Chicago, IL 60612, USA. Received: 23 September 2020; Accepted: 29 October 2020; Published: 30 October 2020 Abstract: Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family.