Materials Wzt
1. 10X DNase digestion buffer 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2. 2. RQ1 RNase-free DNase Promega, Madison, WI . 3. 10X Proteinase K digestion buffer 100 mM Tris-HCl, pH 8.0, 100 mM ethylene diamine tetraacetic acid EDTA , 2.5 w v sodium dodecyl sulfate SDS . 4. Proteinase K, PCR grade Roche Applied Sciences, Indianapolis, IN . 5. Wizard DNA Clean-up System Promega, Madison, WI . 6. 80 v v Ethanol in RNase- and DNase-free water. 8. 2X SYBR Green PCR Master Mix Applied Biosystems, Foster...
Virus Concentration
These virus concentration methods were 15-19 devised principally for the subgroup C viruses Ad2 and Ad5, but Ad40 is considered to be sufficiently similar so that the methods remain valid. It should be noted that there are discrepancies between the values obtained by the various methods, and it is recommended that for consistency, one method should be chosen and adhered to. 1. Using the PicoGreen dsDNA Quantitation Kit Invitrogen Molecular Probes dilute the stock T E from the kit 1 20 in H2O....
Notes Jcc
1. Test bottles of medium are incubated at 37 C for 5 d to ensure sterility for each batch. Sterility can also be tested on blood agar plates or in nutrient broth. Care should be taken to cover the medium during preparation and to prepare medium in an area not normally used for handling of virus adenovirus virions can pass through a 0.22- m filter . Bottles, a 20-L container, and stir bar used for media preparation should be dedicated for tissue culture use and not mixed with chemical...
Peter Groitl and Thomas Dobner
This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 Ad5 mutants carrying defined mutations in early transcription units 1 E1 and 4 E4 . The strategy involves three recombinant plasmids containing E1 pE1-1235 , E4 pE4-1155 , or the wild-type genome that lacks a portion of E3 pH5 g4100 . To generate recombinant viruses, mutations are first introduced into pE1- and or pE4-transfer plasmids by site-directed mutagenesis. The mutagenized...
Volume 2 Ad Proteins RNA Lifecycle Host Interactions and Phylogenetics
1 Analysis of the Efficiency of Adenovirus Transcription Cristina Iftode and S. J. Flint 2 The Use of In Vitro Transcription to Probe Regulatory Functions of Viral Protein Domains Paul M. Loewenstein, Chao-Zhong Song, and Maurice Green 3 Preparation of Soluble Extracts From Adenovirus Infected Cells for Studies of RNA Splicing Oliver M hlemann and G ran Akusj rvi 4 In Vitro Methods to Study RNA Interference During an Adenovirus- Gunnar Andersson, Ning Xu, and G ran Akusj rvi 5 Simultaneous...
Structural Capsid Proteins Expressed From the Major Late Transcription Unit
Adenovirus infections are temporally defined based on an ordered progression of gene transcription. In the majority of vectors, the immediate early gene E1A and two early regions, E1B and E3, have been deleted. Following early gene expression is the onset of DNA replication, which coincides with late gene expression. All viral capsid proteins are expressed during the late stage of viral infection and essentially all with protein IX being the sole exception are transcribed from the major late...
Plaque Assays on A549 Cells for Determination of Adenovirus Titers see Notes 13
One day prior to plaque assay, A549 cells are plated at 2.0 x 106 cells 60-mm dish Corning, Falcon . 1. On the day of the plaque assay, dishes of confluent A549 cells are washed with 5 mL of serum-free DMEM for 30-60 min prior to addition of the diluted virus this wash medium is removed immediately before the addition of the virus dilutions. 2. Serial dilutions of virus are made in serum-free DMEM dilutions are done within a laminar flow hood. Virus is diluted in sterile disposable snap-cap...
Materials Sfb
1. 10X Tris borate EDTA electrophoresis buffer 108 g Tris base, 55 g boric acid, 4 mL 0.5 M ethylene diamine tetraacetic acid EDTA , pH 8.0, per liter. Store at room temperature. 2. 0.6 Agarose in 1X TBE for gel electrophoresis. 3. Ethidium bromide stock solution 10 mg mL store at 4 C. 4. 1 M Tris-HCl , pH 8.0 autoclave and store at room temperature. 5. Herring sperm DNA dissolve herring sperm DNA in 10 mM Tris-HCl, pH 7.0, and sonicate until the viscosity of the solution no longer changes...
Growth of Monolayer and Suspension Cultures
KB suspension cultures are a clonal line derived in the laboratory of Maurice Green from a KB suspension culture received originally from Dr. Harry Eagle and grown in the laboratories of Maurice Green and William Wold. This clonal line is reported to produce higher yields of virus than the 1. KB cells are grown in suspension in Joklik-modified minimum essential medium MEM 5 heat-inactivated horse serum in Florence boiling flasks Pyrex or Kimax see Note 5 . Cells are maintained in culture with...
Mouse Brain Homogenization
1. For each sample to be homogenized, label an autoclaved 2-mL screw-cap vial that is three-quarters filled with 1.0-mm glass beads see Note 2 . In addition, label two 1.5-mL microfuge tubes for each sample see Note 6 . 2. Tare the labeled 2-mL screw-cap vials and record the weight on the side of vial using an ethanol-resistant marker. 3. Attach a sterile scalpel blade to a sterile scalpel handle, dip in ethanol, and flame to burn the ethanol off. 4. In a biosafety cabinet, cut a small piece of...
DAPI Staining Assay
DAPI staining is often one of the first assays performed for detection of apoptotic cells. This procedure is also described as a dye exclusion method. This means that intact and damaged plasma membranes can be discriminated by staining. DAPI is known primarily for its ability to form fluorescent complexes with natural double-bonded strands of DNA, showing its fluorescence at those areas that contain hydrogen bonds 8,9 . Cells with injured plasma membranes are permeable to the stain, whereas...
Angela N Cauthen Amanda R Welton and Katherine R Spindler
Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wildtype mouse adenovirus, production of viral DNA and viral DNA-protein complex, and trans-fection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of...
Cell Culture Media and Stock Solutions
1. Dulbecco's modified Eagle's medium DMEM with high glucose, with L-glutamine, with phenol red, without sodium pyruvate, without sodium bicarbonate Invitrogen, or JRH Biosciences . 2. Sodium bicarbonate tissue culture grade Invitrogen or Sigma . 3. Penicillin streptomycin stock 1000X 10,000 U mL of penicillin G sodium and 10,000 g mL streptomycin sulfate in 0.85 saline Invitrogen . Store at -20 C. 4. 0.22- im Filters Millipore, Corning, or Nalge . 5. Minimum essential medium S-MEM...
Analysis of the SubGf Subdiploid Population
This analysis allows for the quantification, upon staining, of the percentage of the subdiploid population. Cells with lower DNA staining than that of G1-cells sub-G1 peaks are considered apoptotic 8,22 . The sub-G1 analysis is performed following the rinse and staining processes see Note 6 . 1. Plate 5 x 104 tumor cells in a 35-mm dish. 2. On the next day, infect the cells with AdWTp53 multiplicity of infection MOI of 100 PFU cell for 48 h . 3. Add 1 mL of trypsin and prepare single-cell...
Introducing ts125 Mutation Into pacAd5 92100
We have used in vivo homologous recombination in E. coli 12 to introduce the H5ts125 mutation into pacAd5 9.2-100 according to the following protocol. 1. Digest pacAd5 9.2-100 with BstZ17 I and gel-purify the vector and the attached Ad5 sequences there are two sites for BstZ171 in the Ad5 genome at nucleotide positions 5764 and 29010 and no site in the vector . 2. Take OD and calculate the amount of vector. 3. Dilute the digested vector to a concentration of 1 g mL in TE buffer. 4. Dilute...
Preparation of Viral DNA From Infected Cells Modified Hirt Extraction 13
a. If the cells are firmly adhering, wash in TBS, drain, add 0.6 mL of Hirt buffer, incubate for 10 min at room temperature, and scrape lysate into Eppendorf tube. b. If cells are detaching from plate, scrape cells into medium, centrifuge at 2000g for 10 min or at 15,000g for 1 min, resuspend cell pellet in 0.6 mL Hirt buffer, transfer to Eppendorf tube, and incubate for 10 min at room temperature. 2. Add 0.15 mL 5 M NaCl mix gently by inverting tube. 4. Centrifuge at 15,000g for 30-45 min. 5....
Vivien Mautner
The enteric adenoviruses of subgroup F Ad40 and Ad41 pose some special problems of cultivation, as they cannot be readily passaged in many of the cell types used to propagate the more commonly used subgroup C serotypes Ad2 and Ad5 and there is no standard plaque assay. Methods to propagate Ad40 in complementing cell lines and to evaluate infectivity and particle number are presented in this chapter. Key Words Enteric adenovirus type 40 fluorescent focus assay Hirt extraction virus particle...
Index
Adenovirus death protein ADP , 232 Adenovirus Ad type 40. See Enteric Ad40 Adenovirus vectors. See Vectors Animal models. See Cotton rat, Syrian hamster Antigen-capture ELISA, 218-219 capture ELISA, 215 MAV-1 infected organs, 215 mouse brain homogenization, 217-218 optimization, 219 plaque assay, 215 quantitation, 215 titration, 215 virus detection, 215 Apoptosis, 135, 136, 137. See also p53, shRNA vectors Apoptosis, cellular genes in virus- induced. See shRNA vectors Atadenovirus. See Ovine...
Fluorescent Focus Assay
1. Set up monolayers of cells in 35-mm plates or Linbro wells at 3 x 104 cells well. 3. Infect with 0.1 mL virus dilution in TBS. 4. Adsorb virus for 90 min at 37 C, shaking plates every 20-25 min. 5. Overlay with DMEM 0.5 FCS. 7. Remove medium wash twice with complete PBS. Inspect cells to confirm morphology and adherence. 8. Fix with ice-cold 90 methanol for 4 min. 9. Wash twice with PBS. Plates may now be stored at 4 C with 1 mL of PBS overlay. 10. Add antibody at dilutions 1 50, 1 150, 1...
Materials Pck
1. QIAGEN Plasmid Maxi Kit QIAGEN, Mississauga, ON, Canada, cat. no. 2. QIAprep Spin Miniprep Kit QIAGEN, Mississauga, ON, Canada, cat. no. 3. Pfu DNA polymerase with 10X Pfu buffer Stratagene, La Jolla, CA . 4. Polymerase chain reaction PCR tubes 0.2-mL VWR, cat. no. 20170-010 . 5. Premium flex Eppendorf 1.5-mL tubes VWR, cat. no. CA20901-551 . 7. Ultrapure agarose Invitrogen, cat. no. 15510-027 . 8. QIAquick gel extraction kit QIAGEN, Mississauga, ON, Canada, cat. no. 28704 . 9. Chemically...
Making a Recombinant Adenovirus Containing a New Terminal Exon in the MLTU
Two methods are described for generating a recombinant virus the first uses recombination in bacterial cells 4,5 . The second procedure is direct recombination in complementing 293 cells 6 . The principles behind both strategies are similar. The general strategy relies on cotransfection of a linearized pAdCiG viral vector recipient with a linearized pAd70-100 F X modified plasmid donor Fig. 4 . In bacteria, through double-recombination between regions of homology and kanamycin selection, a...
Ann E Tollefson Mohan Kuppuswamy Elena V Shashkova Konstantin Doronin and
Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus either replication-competent or replication-defective in spinner cultures e.g., KB, HeLa, or 293 cells . Protocols for harvesting...
Contributors
G. Eric Blair, PhD School of Biochemistry and Molecular Biology, University of Leeds, Leeds, United Kingdom Gerald W. Both, PhD CSIRO Molecular and Health Technologies, North Ryde, New South Wales, Australia Graham Bottley, msc, PhD School of Biochemistry and Molecular Biology, University of Leeds, Leeds, United Kingdom Julie Boyer, PhD Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY Fiona Cameron, PhD CSIRO Molecular and Health Technologies, North...
Julie Boyer and Gary Ketner
Adenovirus early region 4 E4 regulates processes in infected cells that include viral late gene expression, nonhomologous end joining, responses to DNA damage, and apoptosis. E4 is essential for viral growth in most cell lines. In this chapter, the current knowledge of the functions of six E4 products is summarized briefly. Protocols are presented for manipulation of E4, incorporation of E4 mutations into the viral genome, and growth of E4 mutants on complementing cell lines. A compilation of...