DOCSLIB.ORG

Regulation of Transgene Expression in Genetic Immunization

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Transgene Expression in Genetic Immunization Brazilian Journal of Medical and Biological Research (1999) 32: 155-162 Regulation of transgene expression 155 ISSN 0100-879X Regulation of transgene expression in genetic immunization J.S. Harms1, 1Department of Animal Health and Biomedical Sciences, University of Wisconsin, S.C. Oliveira2 Madison, WI, USA and G.A. Splitter1 2Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil Abstract Correspondence The use of mammalian gene expression vectors has become increas- Key words J.S. Harms ingly important for genetic immunization and gene therapy as well as · DNA vaccine AHABS basic research. Essential for the success of these vectors in genetic · Gene therapy 1655 Linden Drive · immunization is the proper choice of a promoter linked to the antigen Regulation Madison, WI 53706 · Transcription of interest. Many genetic immunization vectors use promoter elements USA · Transgene Fax: +1-608-262-7420 from pathogenic viruses including SV40 and CMV. Lymphokines E-mail: [email protected] produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promot- Research supported by the Robert ers. Our objective was to determine the effect of IFN-g on transgene Draper Technology Innovation Fund expression driven by viral SV40 or CMV promoter/enhancer and the (No. 135-0546) of the University mammalian promoter/enhancer for the major histocompatibility com- of Wisconsin, Madison. plex class I (MHC I) gene. We transfected the luciferase gene driven Presented at the International by these three promoters into 14 cell lines of many tissues and several Symposium “The Third Revolution species. Luciferase assays of transfected cells untreated or treated with g on Vaccines: DNA Vaccines”, IFN- indicated that although the viral promoters could drive lu- Belo Horizonte, MG, Brasil, ciferase production in all cell lines tested to higher or lower levels than November 3-7, 1997. the MHC I promoter, treatment with IFN-g inhibited transgene expres- sion in most of the cell lines and amplification of the MHC I promoter- driven transgene expression in all cell lines. These data indicate that Received October 5, 1998 the SV40 and CMV promoter/enhancers may not be a suitable choice Accepted November 3, 1998 for gene delivery especially for genetic immunization or cancer cy- tokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system. Introduction way, proteins produced through mammalian expression vectors can stimulate humoral or During the past few years, the use of cellular immunity. Intracellular expression mammalian gene expression vectors for new of a non-secreted antigen should specifically methods such as genetic immunization has induce cellular immunity through presenta- become increasingly important. Recent suc- tion by the major histocompatibility com- cess with DNA vaccines indicates their enor- plex class I (MHC I) molecules to cytotoxic mous future potential in diverse fields such T cells. During the immune response, many as infectious diseases, allergy and cancer (1- lymphokines are produced by the various 6). Depending on the antigen processing path- cells involved, including IFN-g, that can act Braz J Med Biol Res 32(2) 1999 156 J.S. Harms et al. on the target cell carrying the transgene. public concern over transfecting DNA ele- Modulation of expression of the transfected ments from pathogenic or tumor-causing vi- gene could occur and, depending on the ruses. BL3-6prmtr is the promoter for the promoter used, increase or decrease trans- cattle major histocompatibility complex class gene expression. Therefore, the proper choice I gene. MHC I is expressed on nearly all of promoter/enhancer linked to the gene of tissues, is critical in immune system commu- interest is critical to the success of DNA nication, and can be up-regulated by lym- vaccinations. phokines such as IFN-g (9). We used the mammalian MHC I pro- The SV40, CMV, and MHC I promoter/ moter/enhancer, BL3-6prmtr (7), and com- enhancers have complex cis elements that pared transgene expression and effects of bind varied cellular trans-factors (10-12). IFN-g regulation to the popular viral pro- SV40 and MHC I promoters both have AP2 moter/enhancers of SV40 and CMV in vari- binding sites while CMV and MHC I pro- ous cell lines of several species. To date, moters both have ATF binding sites. In com- almost all commercial mammalian expres- mon to all three promoters are AP1 and sion vectors use viral promoter/enhancer se- NFkB binding sites. Additionally, all three quences from pathogenic viruses such as promoters have interferon response se- simian virus 40 (SV40), or human cytome- quences. However, for CMV and SV40, fac- galovirus (CMV). Although these promoter tors binding to this region can inhibit tran- elements are from pathogenic viruses, they scription (13,14), whereas, for MHC I, inter- have become very useful due to high tran- feron response factors enhance transcription scription initiation ability in most mamma- (15,16). lian tissues (8). However, in genetic immu- nization applications involving human pa- Comparison of luciferase expression tients or animal husbandry, a mammalian driven by MHC I, SV40, and CMV promoter may be more desirable for easing promoter/enhancers Table 1 - Cell lines used for transfections. The luciferase gene driven by BL3-6prmtr (cattle MHC I), SV40, or CMV promoter/ All cell lines were provided by the American Type Culture Collection (ATCC). enhancer was used as a reporter gene tran- siently transfected in cell lines of various Species Cell line Tissue tissues from several species (listed in Table Mouse A20.J B lymphoma 1). Assays of luciferase production were C2C12 Myoblast used to compare the relative strengths of the G8 Myoblast MHC I promoter/enhancer to the viral pro- P815 Mastocytoma moter/enhancers transgene expression abili- RAW 264.7 Macrophage ties in each cell line. Each of the three pro- Human COLO 320HSR Colon carcinoma moters was able to drive luciferase expres- LCL 721.221 B lymphoblast SW 837 Rectal carcinoma sion in all cell lines tested. However, BL3- 6prmtr was, in many cases, weaker than Cow BL3.1 B sarcoma MDBK Kidney either SV40 or CMV promoters. The data, summarized in Table 2, show that SV40 was Dog D17 Osteocarcinoma stronger than BL3-6prmtr in 12/14 cell lines Monkey Vero Kidney and CMV was stronger in 6/14 cell lines. Rat NMU Mammary carcinoma Comparisons between SV40 and CMV pro- Sheep FLK Kidney moter/enhancers could not be made because the vector constructs were not similar. Braz J Med Biol Res 32(2) 1999 Regulation of transgene expression 157 Effect of IFN-g addition to transient Table 2 - Effect of IFN-g on luciferase transgene production driven by MHC I, SV40, or transfected cell culture on transgene CMV promoters. promoter Note: Increased expression after IFN-g treatment is in bold numbering. Decreased expression after IFN-g treatment is in italics numbering. aThree transfections per cell Luciferase production was positively or line of each luciferase gene construct (in parentheses) driven by MHC I, SV40, or CMV negatively altered by IFN-g treatment de- promoters were done for each experiment. Transfections were performed using the cationic lipid method (LipofectAMINE; Life Technologies, Inc., Gaithersbourg, MD). At pending on the promoter and cell line. Tran- least three experiments were done for each cell line. Transfection efficiency was siently transfected cell cultures were treated measured using internal controls for either ß-galactosidase (pGL6 and pGL2 Control) or g CAT (p6/Luc and pcDNA3/Luc). bIFN-g was added (+) or not added (-) to transient or not treated with rIFN- for 16 h and then transfects which were assayed for luciferase production 16 h later. Human rIFN-g was assayed for luciferase production. Results added to human, cow, dog, monkey, and sheep cell lines while mouse rIFN-g was demonstrate that BL3-6prmtr-driven lu- added to mouse and rat cell lines (see Table 1). Numbers represent mean picogram (pg) luciferase per transfection calculated using a luciferase standard curve. The ciferase production increased in all cell lines, coefficient of variation was less than 10% for each experiment. whereas the SV40 or CMV promoter-driven luciferase production decreased in most cell Cell line MHC I SV40 CMV lines (Table 2). (pGL6)a (p6/Luc) (pGL2 Control) (pcDNA3/Luc) Using the mammalian expression vectors b p6/Luc (MHC I promoter driven) and (-) (+) (-) (+) (-) (+) (-) (+) pcDNA3/Luc (CMV promoter driven), we A20.J 30 41 50 81 826 409 126 16 established stable transfects of several cell BL3.1 40 54 7 10 8 5 4 2 52 61 g C2C12 19 20 403 83 182 87 lines to ascertain whether IFN- would af- COLO 56 107 30 53 28 11 11 2 fect chromosome integrated promoter/trans- D17 38 40 14 16 88 68 30 22 gene expression differently than episomal FLK 36 251 99 234 215 354 59 89 G8 28 96 9 17 810 209 53 23 transgene expression. We isolated neomy- 721.221 34 39 19 20 43 18 18 17 cin-resistant clones of A20.J, P815, RAW MDBK 8 9 8 10 10 9 13 8 NMU 24 63 91 176 250 75 154 34 264.7, COLO 320HSR, SW 837, and BL3.1 P815 28 30 4 5 57 34 4 2 cell lines and selected the highest luciferase- RAW 20 74 12 25 50 176 9 11 expressing clones of each transgene con- SW837 8 9 9 10 16 12 8 7 Vero 34 62 14 35 208 150 10 7 struct. IFN-g treatment of these stable trans- fects produced similar results (Table 2) us- g ing the same IFN- dosage (500 U/ml) and C2C12 G8 measurement time point (16 h; data not 180 450 shown).
Load more

Recommended publications
  • Gene Therapy Glossary of Terms
    GENE THERAPY GLOSSARY OF TERMS A • Phase 3: A phase of research to describe clinical trials • Allele: one of two or more alternative forms of a gene that that gather more information about a drug’s safety and arise by mutation and are found at the same place on a effectiveness by studying different populations and chromosome. different dosages and by using the drug in combination • Adeno-Associated Virus: A single stranded DNA virus that has with other drugs. These studies typically involve more not been found to cause disease in humans. This type of virus participants.7 is the most frequently used in gene therapy.1 • Phase 4: A phase of research to describe clinical trials • Adenovirus: A member of a family of viruses that can cause occurring after FDA has approved a drug for marketing. infections in the respiratory tract, eye, and gastrointestinal They include post market requirement and commitment tract. studies that are required of or agreed to by the study • Adeno-Associated Virus Vector: Adeno viruses used as sponsor. These trials gather additional information about a vehicles for genes, whose core genetic material has been drug’s safety, efficacy, or optimal use.8 removed and replaced by the FVIII- or FIX-gene • Codon: a sequence of three nucleotides in DNA or RNA • Amino Acids: building block of a protein that gives instructions to add a specific amino acid to an • Antibody: a protein produced by immune cells called B-cells elongating protein in response to a foreign molecule; acts by binding to the • CRISPR: a family of DNA sequences that can be cleaved by molecule and often making it inactive or targeting it for specific enzymes, and therefore serve as a guide to cut out destruction and insert genes.
    [Show full text]
  • Lecture 3 Types, Biology and Salient Features of Vectors in Recombinant
    NPTEL – Bio Technology – Genetic Engineering & Applications MODULE 1- LECTURE 3 TYPES, BIOLOGY AND SALIENT FEATURES OF VECTORS IN RECOMBINANT DNA TECHNOLOGY – PLASMID 1-3.1 Introduction: DNA molecule used for carrying an exogenous DNA into a host organism and facilitates stable integration and replication inside the host system is termed as Vector. Molecular cloning involves series of sequential steps which includes restriction digestion of DNA fragments both target DNA and vector, ligation of the target DNA with the vector and introduction into a host organism for multiplication. Then the fragments resulted after digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. In general, vectors should have following characteristics: • Capable of replicating inside the host. • Have compatible restriction site for insertion of DNA molecule (insert). • Capable of autonomous replication inside the host (ori site). • Smaller in size and able to incorporate larger insert size. • Have a selectable marker for screening of recombinant organism. 1-3.2 Plasmids: • Plasmids are naturally occurring extra chromosomal double-stranded circular DNA molecules which can autonomously replicate inside bacterial cells. Plasmids range in size from about 1.0 kb to over 250 kb. • Plasmids encode only few proteins required for their own replication (replication proteins) and these proteins encoding genes are located very close to the ori. All the other proteins required for replication, e.g. DNA polymerases, DNA ligase, helicase, etc., are provided by the host cell.Thus, only a small region surrounding Joint initiative of IITs and IISc – Funded by MHRD Page 28 of 84 NPTEL – Bio Technology – Genetic Engineering & Applications the ori site is required for replication.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • RNA Viruses As Tools in Gene Therapy and Vaccine Development
    G C A T T A C G G C A T genes Review RNA Viruses as Tools in Gene Therapy and Vaccine Development Kenneth Lundstrom PanTherapeutics, Rte de Lavaux 49, CH1095 Lutry, Switzerland; [email protected]; Tel.: +41-79-776-6351 Received: 31 January 2019; Accepted: 21 February 2019; Published: 1 March 2019 Abstract: RNA viruses have been subjected to substantial engineering efforts to support gene therapy applications and vaccine development. Typically, retroviruses, lentiviruses, alphaviruses, flaviviruses rhabdoviruses, measles viruses, Newcastle disease viruses, and picornaviruses have been employed as expression vectors for treatment of various diseases including different types of cancers, hemophilia, and infectious diseases. Moreover, vaccination with viral vectors has evaluated immunogenicity against infectious agents and protection against challenges with pathogenic organisms. Several preclinical studies in animal models have confirmed both immune responses and protection against lethal challenges. Similarly, administration of RNA viral vectors in animals implanted with tumor xenografts resulted in tumor regression and prolonged survival, and in some cases complete tumor clearance. Based on preclinical results, clinical trials have been conducted to establish the safety of RNA virus delivery. Moreover, stem cell-based lentiviral therapy provided life-long production of factor VIII potentially generating a cure for hemophilia A. Several clinical trials on cancer patients have generated anti-tumor activity, prolonged survival, and
    [Show full text]
  • Clone the Unclonable – Vectors and Cells to Capture and Express Problematic Dnas
    Clone the Unclonable – Vectors and Cells to Capture and Express Problematic DNAs Ron Godiska, Ph.D. May, 2016 Agenda Improve Cloning and Expression of Problematic DNA • What makes DNA difficult to clone or express? • Circular vectors for difficult DNAs • Linear vectors for “impossible” DNAs • Enzyme-free cloning and compatible protein expression vectors • E. coli strains for toxic recombinant proteins or unstable DNA What is Unclonable DNA? Characteristics of Difficult DNA • Toxic coding sequences • Promoters • A-T Rich DNA • Large fragments (>10 kb) • Trace amounts Which types of difficult DNA have you worked with in the lab? Common Vector Traits Cause Cloning Problems Vector driven transcription into the insert – Deleterious expression – Destabilize 2o structure Insert driven transcription out into the vector – False negatives (blue or lethal) – Transcriptional interference pUC19 – Replication interference High copy number – Increased effect of the above issues – Active origin – Many copies/cell Supercoiling – Deletion of secondary structures CloneSmart® Technology “Silencing” DNA Inserts to Improve Cloning • No Transcriptional Interference – Stably maintain “unclonable” DNA • No blue/white screening – No false positives/negatives • Minimal vector size • Variable copy number • Variety of drug selection markers • Very low empty-vector background U.S. Patent 6,709,861 pSMART Vectors Clone Toxic Inserts and Promoters Test DNA Fragments: • RNase coding sequence (350 bp; no promoter) • Phage Lambda PR Promoter (400 bp) pSMART Vectors cDNA Clones are More Stable Deletion Rates: <1% in pSMART HC Kan vs. 32% in pUC % Deleted Clones Insert Size (kb) • Teleost fish cDNA library • Clones were grown in liquid medium, diluted, and regrown • Each final culture was diluted, plated, and grown up into individual colonies • Plasmid DNA was isolated from colonies for each clone and analyzed - Oleksiak M.F., Crawford D.L., 2001.
    [Show full text]
  • Important Vector Factors for Gene Expression Technical Reference Guide
    BioResearch Important Vector Factors for Gene Expression Technical Reference Guide vectors expressing the same luciferase gene in different backbones and expression cassettes and obtained highly discriminative expres- sion levels. In this guideline, we show the most important aspects to be considered when choosing an expression vector and their impact on your experimental results. 1. Promoter Strength Introduction Is your promoter appropriate for the cell type that you are working with? Our Scientific Support Team is commonly asked: “Why don’t I get the Table 1 describes the promoter strengths as a relative percent of the same expression level of my gene if I use various vector backbones?” strength of the CMV promoter for various cells for which Lonza has There are many components to a vector that can have an effect on the Optimized Protocols. The CMV promoter activity is set to 100% based level of gene expression. Below you will find the 10 most important on their CAT assay values from the referenced publications. Although factors to consider when looking at vectors. CMV is a strong promoter in many mammalian cells, another promoter The selection of an appropriate expression vector is crucial for efficient may give stronger expression in your cells (e.g., promoter SV40 in gene expression. Just take a look at Figure 1. We tried 10 different BHK-21 cells). n 1600 1400 1200 1000 800 [% of pGL3-CMV at 24 h] 24 at pGL3-CMV [% of 600 400 Relative luciferase-expressio Relative 200 0 Control Program Only pGL3-Control pGL3-CMV pmax Cloning™ pSG5 pcDNA3.1 pFP pIRES-MCS B pcDNA4 HisMax pCMVbeta pCMV-TNT 4 hours 24 hours 48 hours n 2000 1500 1000 500 0 Control Program Only pGL3-Control pGL3-CMV pmax Cloning™ pSG5 pcDNA3.1 pFP pIRES-MCS B pcDNA4 HisMax pCMVbeta pCMV-TNT [% of pGL3-CMV at 24 h] 24 at pGL3-CMV [% of 4 hours 24 hours 48 hours Relative luciferase-expressio Relative Figure 1: Luciferase expression levels depend on vector backbones.
    [Show full text]
  • Cloning Technologies for Protein Expression and Purification
    Cloning technologies for protein expression and purification James L Hartley Detailed knowledge of the biochemistry and structure of expedite the manipulations that are often required to individual proteins is fundamental to biomedical research. To obtain pure, active recombinant proteins. further our understanding, however, proteins need to be purified in sufficient quantities, usually from recombinant sources. Cloning for protein expression and Although the sequences of genomes are now produced in purification automated factories purified proteins are not, because their The required amount, purity, homogeneity, and activity behavior is much more variable. The construction of plasmids of the protein of interest usually determine the choice of and viruses to overexpress proteins for their purification is often expression context, vector and host. Usually the first tedious. Alternatives to traditional methods that are faster, easier consideration is what host cell to use. Expression in and more flexible are needed and are becoming available. Escherichia coli is fast, inexpensive and scaleable, and minimal post-translational modifications make proteins Addresses Protein Expression Laboratory, Research Technology Program, purified from E. coli relatively homogeneous and highly SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, USA desirable for structural studies. However, in our experi- ence most eukaryotic proteins larger than 30 kDa are Corresponding author: Hartley, James L ([email protected]) unlikely to be properly folded when expressed in E. coli (JL Hartley et al., unpublished). Expression in mamma- Current Opinion in Biotechnology 2006, 17:359–366 lian cells is best for activity and native structure (includ- ing post-translational modifications), but yields are much This review comes from a themed issue on lower and costs are high.
    [Show full text]
  • Complete Control Retroviral Inducible Mammalian Expression System
    Complete Control Retroviral Inducible Mammalian Expression System Instruction Manual Catalog #217564 (pFB-ERV), #240028 (pCFB-EGSH) Revision D.0 For Research Use Only. Not for use in diagnostic procedures. 217564-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES Email [email protected] World Wide Web www.genomics.agilent.com Telephone Location Telephone United States and Canada 800 227 9770 Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK/Ireland 0845 712 5292 All Other Countries Please visit www.agilent.com/genomics/contactus Complete Control Retroviral Inducible Mammalian Expression System Materials Provided .............................................................................................................................. 1 Storage Conditions .............................................................................................................................. 1 Additional Materials
    [Show full text]
  • An Episomal Expression Vector for Screening Mutant Gene Libraries in Pichia Pastoris
    Plasmid 54 (2005) 80–85 www.elsevier.com/locate/yplas Short communication An episomal expression vector for screening mutant gene libraries in Pichia pastoris Charles C. Lee*, Tina G. Williams, Dominic W.S. Wong, George H. Robertson USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710, USA Received 7 October 2004, revised 6 December 2004 Available online 20 January 2005 Communicated by Manuel Espinosa Abstract Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screen- ing projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the a-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants. Published by Elsevier Inc. Keywords: Pichia pastoris; Episomal expression vector 1. Introduction formed with libraries of randomly mutated genes encoding the enzyme of interest (Cohen et al., There are many efforts directed at improving 2001). Clones that demonstrate an increased activ- enzymes involved in industrial processes in order ity relative to wild type can be isolated and further to increase cost and energy efficiency.
    [Show full text]
  • Retroviral Gene Transfer and Expression User Manual Table of Contents I
    Takara Bio USA, Inc. Retroviral Gene Transfer and Expression User Manual Cat. Nos. Many (081419) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: [email protected] United States/Canada Asia Pacific Europe Japan Page 1 of 31 800.662.2566 +1.650.919.7300 +33.(0)1.3904.6880 +81.(0)77.565.6999 Retroviral Gene Transfer and Expression User Manual Table of Contents I. Introduction ..................................................................................................................................................................... 3 II. List of Components ....................................................................................................................................................... 10 III. Additional Materials Required .................................................................................................................................. 13 IV. Safety Guidelines for Working with Retroviruses .................................................................................................... 16 V. Plasmid Vector Manipulations ...................................................................................................................................... 17 VI. Working with Retroviral Packaging Cell Lines ........................................................................................................ 18 A. General Cell Culture and Retrovirus Information ....................................................................................................
    [Show full text]
  • Advances in the Development of Genetically Modified Animals
    Advances in the Development of Genetically Modified Animals Louis-Marie Houdebinea, Martín Alfredo Lemab, Moisés Burachikc* aBiologie du Développement et Reproduction, INRA, UMR1198, 78350 Jouy en Josas, France bNational University of Quilmes. Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires Province, Argentina cINDEAR (Agrobiotechnology Institute Rosario), Ocampo 210 Bis (Predio CCT), (2000) Rosario, Argentina *E-mail: [email protected] (corresponding author) Abstract A wide range of genetically modified (GM) animal/trait combinations are being developed with a great variety of purposes: to enhance food production or nutritional quality; to improve human or animal health; as sources of products (pharmaceuticals) or tissues (transplantation) for human therapeutic use; to control vectors of human infectious diseases; and to improve animal farming operations, amongst others. This review will focus on developments in three main animal classes: mammals, fish and insects. Several developments of GM mammals and their derived products shall be reviewed as examples of the potential impacts of this technology on farming practices through to human health. Fish, on the other hand, pose a particular problem because of the possible effects of their entry into natural ecosystems as a result of their unintended escape from fish farming operations. Two main objectives drive the development of GM insects, namely pest management and control of vector-borne infectious diseases. These will require the deliberate release of GM insects directly
    [Show full text]
  • Construction of a High-Expression System in Bacillus Through Transcriptomic Profiling and Promoter Engineering
    microorganisms Article Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering Cui-Cui Miao 1, Lin-Li Han 1, Yan-Bing Lu 1 and Hong Feng 1,2,* 1 Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, 29, Wangjiang Rd, Chengdu 610064, China; meff[email protected] (C.-C.M.); [email protected] (L.-L.H.); [email protected] (Y.-B.L.) 2 Key Laboratory for Bio-Resources and Eco-Environment of Ministry of Education, Sichuan University, 29 Wangjiang Rd, Chengdu 610064, China * Correspondence: [email protected]; Tel.: +86-28-8547-0226 Received: 24 May 2020; Accepted: 9 July 2020; Published: 12 July 2020 Abstract: Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B.
    [Show full text]