Direct and Efficient Transfection of Mouse Neural Stem Cells And
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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. -
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 -
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. -
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. -
Improving Electrotransfection Efficiency by Post-Pulse Centrifugation
Gene Therapy (1999) 6, 364–372 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Improving electrotransfection efficiency by post-pulse centrifugation LH Li1,2, P Ross1 and SW Hui1 1Membrane Biophysics Laboratory, Roswell Park Cancer Institute, Buffalo, NY, USA We have demonstrated that the viability of electro- dorf desktop centrifuge. Pelleting improves the cell viability transfected adherent CHO and suspended NK-L, K-562, over the whole range of the NK-L, K-562, L1210 and MC2 L1210 and MC2 cells is improved if pelleting by centrifug- cell concentrations studied. When this pelleting method is ation is performed immediately after pulsing. The protec- applied to load CHO cells with FITC-dextran (41 000 MW), tion effect on cell viability is cell line- and pellet thickness- not only is the success rate close to 100%, but the growth dependent. For forming CHO cell pellets, centrifugation rate is similar to the control, which is far better than the force (300–13 000 g) and duration are not crucial; about conventional electroporation method. Furthermore, the five to 10 cell layers in the pellet provide the optimal protec- transfection efficiency of the five cell lines in pellet is sig- tion effect. NK-L, K-562, L1210 and MC2 cell pellets are nificantly higher than that in suspension. optimally formed by centrifugation at 13 000 g in an Eppen- Keywords: centrifugation pellet; viability; electroporation; electroloading; electrotransfection Introduction method that can improve cell viability is likely to improve electrotransfection efficiency. Electroporation has become a popular method in biotech- Pelleting by centrifugation effectively reduces extra- nology for transferring genetic materials, as well as other cellular volume between cells and leads to reduced biochemicals, such as foreign proteins and drugs, into erythrocyte electrohemolysis by restricting post-pulse cell 1–3 cells. -
Biopolymeric Materials Used As Nonviral Vectors: a Review
Review Biopolymeric Materials Used as Nonviral Vectors: A Review Jailson de Araújo Santos 1 , Daniel Barbosa Liarte 2, Alessandra Braga Ribeiro 3, Marcia dos Santos Rizzo 1 , Marcília Pinheiro da Costa 1 , Josy A. Osajima 1 and Edson C. Silva-Filho 1,* 1 Materials Science and Engineering Graduate Program, UFPI, Teresina, 64049-550 Piauí, Brazil; [email protected] (J.d.A.S.); [email protected] (M.d.S.R.); [email protected] (M.P.d.C.); [email protected] (J.A.O.) 2 Biology Department, UFPI, Teresina, 64049-550 Piauí, Brazil; [email protected] 3 CBQF—Centre of Biotechnology and Fine Chemistry, Associate Laboratory, Faculty of Biotechnology, Catholic University of Portugal, 4169-005 Porto, Portugal; [email protected] * Correspondence: edsonfi[email protected] Abstract: Bacterial transformation and gene transfection can be understood as being the results of introducing specific genetic material into cells, resulting in gene expression, and adding a new genetic trait to the host cell. Many studies have been carried out to investigate different types of lipids and cationic polymers as promising nonviral vectors for DNA transfer. The present study aimed to carry out a systematic review on the use of biopolymeric materials as nonviral vectors. The methodology was carried out based on searches of scientific articles and applications for patents published or deposited from 2006 to 2020 in different databases for patents (EPO, USPTO, and INPI) and articles (Scopus, Web of Science, and Scielo). The results showed that there are some deposits of patents regarding the use of chitosan as a gene carrier. -
Transfection of Human Lymphoblastoid Cells with Herpes Simplex Viral DNA (Epstein-Barr Virus/B Lymphocytes/Mitogens/Infectious Centers) GEORGE MILLER*T, P
Proc. Nati. Acad. Sci. USA Vol. 76, No. 2, pp. 949-953, February 1979 Medical Sciences Transfection of human lymphoblastoid cells with herpes simplex viral DNA (Epstein-Barr virus/B lymphocytes/mitogens/infectious centers) GEORGE MILLER*t, P. WERTHEIMt, G. WILSON*, J. ROBINSON*, J. L. M. C. GEELENt, J. VAN DER NOORDAAt, AND A. J. VAN DER EB§ *Laboratorium voor de Gezondheidsleer, University of Amsterdam, The Netherlands; §Laboratory for Physiological Chemistry, University of Leiden, The Netherlands; *Department of Pediatrics and Epidemiology, Yale University School of Medicine; and tHoward Hughes Medical Institute Laboratory at Yale University School of Medicine, New Haven, Connecticut 06510 Communicated by Dorothy M. Horstmann, November 14,1978 ABSTRACT The "calcium/dimethyl sulfoxide shock" the pellets and centrifuged for 3 hr at 18,000 rpm at 4°C in the method of transfection was adapted for use in human lymphoid Beckman 19 rotor. The pellets were resuspended in 1/200th cell cultures. One microgram of herpes simplex virus type 1 DNA regularly initiated virus replication in four lymphoblastoid cell vol of 0.15 M NaCI/0.05 M Tris, pH 7.4 (TS buffer) containing lines. Per 105 cells exposed to 1 pg of DNA, 0.5-5 cells formed 0.25 M sucrose and placed on top of a continuous 10-50% an infectious center. The minimal infective dose of DNA was (wt/vol) sucrose gradient in TS buffer. The gradient was cen- approximately 500 ng. trifuged in the SW 27 rotor for 1.5 hr at 23,000 rpm at 4°C. The virus band was harvested and dialyzed overnight against TS A reliable method for transfection of human lymphocytes by buffer. -
Non-Viral in Vitro Gene Delivery: It Is Now Time to Set the Bar!
pharmaceutics Review Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar! 1, 1,2, 2 1, Nina Bono y , Federica Ponti y, Diego Mantovani and Gabriele Candiani * 1 GenT Lab, Department of Chemistry, Materials and Chemical Engineering “G. Natta”, Politecnico di Milano, 20131 Milan, Italy; [email protected] (N.B.); [email protected] (F.P.) 2 Laboratory for Biomaterials and Bioengineering, Canada Research Chair I in Biomaterials and Bioengineering for the Innovation in Surgery, Department of Min-Met-Materials Engineering & Research Center of CHU de Quebec, Division of Regenerative Medicine, Laval University, Quebec City, QC G1V 0A6, Canada; [email protected] * Correspondence: [email protected]; Tel.: +39-02-2399-3181 These authors equally contributed to this work. y Received: 3 February 2020; Accepted: 19 February 2020; Published: 21 February 2020 Abstract: Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. -
Agrobacterium-Mediated Transformation of the Genome-Sequenced Poplar Clone, Nisqually-1 (Populus Trichocarpa)
Color profile: Disabled Composite Default screen Plant Molecular Biology Reporter 22: 1–9, September 2004 © 2004 International Society for Plant Molecular Biology. Printed in Canada. Publish by Abstract Agrobacterium-Mediated Transformation of the Genome-Sequenced Poplar Clone, Nisqually-1 (Populus trichocarpa) CAIPING MA, STEVEN H. STRAUSS, and RICHARD MEILAN* Department of Forest Science, Oregon State University, Corvallis, OR 97331-5752 Abstract. The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood (Populus trichocarpa). To im- prove its utility for functional genomics research, having an efficient means for transfor- mation and regeneration is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants with a strain of Agro- bacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was in- corporated in the selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during the early stages of shoot induction. Histochemical assays revealed expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol, transgenic plants were recovered within 6 mo at an effi- ciency of 6%, adequate to produce a large number of transgenic events with modest effort. Full text†: This manuscript, in detail, is available only in the electronic version of the Plant Molecular Biology Reporter.