Growth of Subcutaneous Tumors in the Syrian Hamster

Expand the cells in multiple 150-mm diameter dishes. The number of dishes needed will depend on the number of tumors desired (see Note 11). 2. When dishes are confluent, remove the medium and trypsinize the cells (see Subheading 3.1.1.). For each dish, add 5 mL of trypsin and, when the cells are detached, add 5 mL of medium. Combine the cells from all of the dishes (10 mL per dish) into one tube (i.e., 250-mL tube, if 20 dishes). 3. Determine the cell concentration by performing a cell count on...

Materials

50 PEG (PEG8000, Sigma P-2139) weigh out 250 g of PEG and add water to 500 mL. Stir until dissolved and autoclave. Mix while cooling to prevent precipitation of the PEG. Store at room temperature. 3.1 M Tris-HCl, pH 8.0. Autoclave and store at room temperature. 4. 13-mL Ultracentrifuge tubes (Seton 7030). 5. 1 M Tris-HCl, pH 7.4. Autoclave and store at room temperature. 6. CsCl p 1.2 g cm3 22.49 26.99 g 100 mL of 50 mM Tris-HCl, pH 7.4. Store at room temperature. 7. CsCl p 1.4 g cm3 38.60 54.04...

Intravenous Injection of Ad in Syrian Hamsters

Intravenous injection of virus may be desired to determine the maximum tolerated dose or the efficacy of an oncolytic adenovirus vector when administered systemically. In our experience, the jugular vein is the preferred route for intravenous injection in the hamster. The vessel is easily accessible and using this route of administration poses little risk of accidental death or injury to the animal. 1. First dilute the oncolytic vector to the desired concentration(s) in PBS for the evaluation...

Viral Cloning Vector pAd5 70100

We use the plasmid pAd70-100dlE3 (Fig. 3) for carrying out virus constructions. This plasmid shuttle system is compatible with recombination strategies used in bacteria as well as those used in mammalian cell lines. The essential features of the shuttle vector are highlighted in Fig. 3. This plasmid contains a modified Ad5 fiber terminal exon. The vector contains a unique PacI restriction site upstream of the fiber splice acceptor such that all of the accessory splicing sequences have been...

Construction of Ad5 Recombinants

To generate Ad5 mutants carrying defined mutations in the E1B and E4 genes, point mutations are first introduced into pE1-1235 and pE4-1155 by site-directed mutagenesis (In Vitro Site-Directed Mutagenesis Kit, STRATAGENE) with oligonucleotide primers. 2. Verify mutations from purified plasmids by DNA-sequencing. 3. Digest the mutagenized constructs with the appropriate restriction enzyme(s) pE1-1235 with SwaI BstZ17I and pE4-1155 with BstBI. 4. Digest pH5pg4100 bacmid DNA with the same...

Virus Harvesting and Concentration

This protocol is adapted from a procedure used to isolate human AdV5 from culture medium (14). Except for centrifugation steps, where solutions containing virus are in sealed containers, all work is carried out in a class II biohazard cabinet. 1. After CPE has developed, medium is transferred to conical-bottomed centrifuge tubes (Nalgene 175-mL or Corning 50-mL, depending on the scale of the virus preparation) and floating, intact cells are removed by centrifugation at 1300g for 10 min. Retain...

Real Time Quantitative PCR for Adenovirus Hexon DNA

Quantitative analysis of subgroup C adenovirus DNA from these cellular DNA preparations can now be performed using real-time PCR (8). To assess the presence of adenovirus subgroup C DNA, a sensitive real-time PCR assay was developed using adenovirus hexon-specific primers and a TaqMan probe. Primers were selected from a region that is highly conserved among subgroup C viruses, but is significantly divergent among other subgroups. Primers were modified from those originally described by...

Plaque Purification

Set up MDBK cells in 6-well plates so that they form 60-70 confluent mono-layers the next day. 2. Prepare serial 10-fold dilutions as described in Subheading 3.8.1. 3. Aspirate medium from the wells and add 0.5 mL of the diluted virus (2 wells per each dilution starting from 104). Incubate at 37 C for 1 h. 4. Aspirate virus solution and overlay the cells with agarose as described in Subheading 3.8.2. Add 3 mL of agarose medium mix per well. Incubate at 37 C in a CO2 incubator for 10-14 d. 5....

Notes

To maximize virus yields at harvest, any cells remaining attached to the culture vessel may be recovered by treatment with trypsin, collection in MEM (10 mL), and centrifugation. Cells are combined with residual cells from the medium and lysed with cell lysis buffer (0.25 NP40 in MEM 1.0 mL per roller bottle) on ice for 1 h with occasional vortexing (this occurs while other manipulations are taking place). Centrifuge the combined cell lysate (1600g for 10 min) to pellet the cell debris. Combine...

Injecting Cotton Rats

Subcutaneous injection of cotton rats generally requires two people one to restrain the animal and one to perform the injection. The cotton rat can be restrained using the methods described above. Short-term anesthetic restraint may be used to facilitate animal handling. The injection site(s) should be shaved using Wahl Peanut clippers or other appropriate clippers. The clipper operator should take care not to nick the skin. The shaved site should be swabbed with 70 ethanol prior to injection....

Introdution Adinoviruse

Construction of mouse adenovirus type 1 (MAV-1) mutants has facilitated studies of adenoviral pathogenesis in the natural host. We have isolated viral mutants of MAV-1 early regions 1A (E1A) and 3 (E3) (1-4). These mutants have altered lethality in adult mice, as measured by 50 lethal dose (LD50) assays. E1A mutants have increased sensitivity to type I and type II interferon in vitro relative to wild-type (wt) virus (5). MAV-1 E1A interacts with mouse Sur2, a subunit of mediator complex, and...

Isolation and Identification of Mutants

Picking Plaques and Extracting Viral DNA 1. After plaques have formed on the experimental plates, circle plaques, using an ethanol-soluble marker, and use a plugged Pasteur pipet to pick each of the plaques from the experimental plates first. Then pick one or two plaques from the positive control plate or the background plate. Pipet the agarose plug into 0.5 mL of 1X PBS. Vortex the samples to break up the agarose and distribute the virus. Note In all the following steps, be careful not...

Complementing Cell Lines

E4 mutants lacking both ORF3 and ORF6 are defective for growth in normal adenovirus hosts and must be propagated on cell lines capable of supplying required E4 functions in trans. The most commonly used such cell line is W162 (11). The W162 cell line was constructed by introducing the Ad5 B fragment (map units 86.3 to 100) into Vero cells as part of a plasmid carrying the selectable marker gpt. W162 cells contain intact E4 and supply the E4 functions required to support the growth of deletions...

Flow Cytometric Data Analysis

Display FITC histograms of control and anti-hexon-labeled samples. Set a marker on the uninfected hexon-labeled histogram, with less than 1 of cells being within the marker region, as shown in Fig. 3. Apply this marker region to the infected samples (Fig. 4). Obtain percentage of cells within the marker region. 2. To calculate FFU from the flow cytometric data will require calibration experiments for each individual system. In our system (see Note 5), the percentage of positive cells in the...

Investigate the Ability of an Oncolytic Ad to Spread Cellto Cell in LCRT Cells

Oncolytic Ads are expected to produce progeny virus and infect neighboring cells in the infected tumor. To assess the speed of this infection-virus production-release-reinfection cycle, a virus-spread assay can be performed. In this assay, parallel vessels containing the cell line are infected with various multiplicities (generally ranging from 102 to 10-4 PFU cell) of an oncolytic Ad. The cells infected with high MOI will be lysed within one infectious cycle, whereas multiple virus...

Maria A Thomas Drew L Lichtenstein Peter Krajcsi and William S M Wold

A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the...

Introduction

Recombinant adenoviruses (Ads) are one of the most commonly used viral vectors for a variety of therapeutic and research purposes both in vitro and in vivo. Several attractive features of Ads have made them the vectors of choice for many applications in gene expression, gene therapy, and oncolytic vectors. These attractive features include the following the virus is able to infect a From Methods in Molecular Medicine, Vol. 130 Adenovirus Methods and Protocols, Second Edition, vol. 1...

Preparation of Virus Seed Stock see Note

Ad40 seed stock should be generated in 60-mm plates by serial passage of 1 10 dilutions. This is preferable to using large flasks, as spot contamination of individual plates can be readily recognized and stocks discarded accordingly. 1. Remove medium from subconfluent monolayers of 293 or KB16 cells in 60-mm plates. 2. Infect with virus diluted in TBS to give approx 1 FFU cell in 0.1 mL plate (generally a 1 10 dilution). 3. Adsorb at 37 C for 60-90 min. Rock plates gently at 20-min intervals....

References

Mautner, V., Steinthorsdottir, V., and Bailey, A. (1995) The enteric adenoviruses, in The Molecular Repertoire of Adenoviruses (Doerfler. W. and Boehm P., eds.), Curr. Topics Microbiol. Immunol. 199 III, Springer Verlag, New York, pp. 229-282. 2. Favier, A.L., Schoehn, G., Jaquinod, M., Harsi, C., and Chroboczek, J. (2002) Structural studies of human enteric adenovirus type 41. Virology 293, 75-85. 3. deJong, J. C., Wigand, R., Kidd, A. H., et al. (1983) Candidate adenoviruses 40 and 41...

Culturing LCRT Cells

LCRT cells can be cultured as an adherent monolayer (see Note 1) in DMEM containing L-glutamine and 10 FBS at 37 C in a humidified incubator with 5 CO2 content. 2. Passage the cells every 3 d (when cells reach about 80-90 confluency) at a dilution of 1 3 to 1 6. Remove the medium from the monolayer, and wash it with prewarmed (37 C) PBS. Add prewarmed trypsin-EDTA solution (1 mL for a T-75 flask), and incubate it at 37 C for 5 min (or until all cells are rounded up). Collect the cells by adding...

Real Time PCR

Prepare a master mix for the real-time PCR reaction as listed below. The volumes shown below are for a single reaction. Prepare enough master mix to run each reaction in duplicate (see Note 8) a. 25 L 2X SYBR Green PCR Master Mix. b. 2 L Forward primer (1.25 M working stock). c. 2 L Reverse primer (1.25 M working stock). d. 14 L RNase- and DNase-free water. 2. Dispense 43 L of the real-time PCR master mix into each reaction vessel (see Note 9). 3. Prepare a 1 10 and 1 100 dilution of each...

Preparation of Virus Stocks

Making a Virus Stock From a Plaque 1. Set up one to two wells (24-well plate) of mouse 3T6 cells per purified mutant plaque so that they will be 75-80 confluent at the time of infection. 2. When the cells are ready, infect one or two wells per mutant by adding one-quarter to one-half of the purified plaque stock (see Note 15). Incubate at 37 C for 1 h and add approx 1 mL of prewarmed 1X DMEM containing 1 HICS. Monitor for CPE (see Note 14). 3. When the cells have significant CPE or have...

Recombination in 293 Cells Fiber Shuttle Plasmid and Viral Large Fragment

The production of novel fiber-modified vectors can be carried out by homologous recombination in 293 cells by transfecting a viral large fragment DNA corresponding to the left end of the Ad genome (Fig. 4) with a donor plasmid bearing the fiber-containing right end of the Ad genome (7,8). Viral large fragment can be obtained from plasmid backbones such a pvAdCiG or from viral DNA. A protocol for purifying viral DNA from a virus stock (1012 particles) is described. 3.6.1. Small-Scale...

Handling and Husbandry of Cotton Rats

Caging cotton rats are extremely excitable animals that are difficult to handle. For the convenience of the handler, cotton rats should be single-housed in polycarbonate rat cages, containing a stainless steel wire bar lid that snaps down tightly. Tekfresh rodent bedding is placed in the bottom of the cage for nesting. For environmental enrichment, a 10-cm piece of 3'' PVC pipe is placed in the cage. This pipe gives the animal a place to hide and helps to decrease the animal's stress. 2. Diet...

William S M Wold Ann E Tollefson

Department of Molecular Microbiology and Immunology Saint Louis University School of Medicine St. Louis, Missouri 2007 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of...

Large Scale Adenovirus Preparation

Spinner KB or 293 cells are used for large-scale production of adenoviruses. Cells are grown in MEM, Joklik-modified a suspension medium with reduced calcium and increased levels of phosphate with 5 HS heat-inactivated at 56 C for 30 min to inactivate complement . For infection, it is typical to use a 3- to 6-L volume of cells that have reached a density of 3-3.5 x 105 cells mL. For a 3-L infection, the volume is reduced to 1 L by centrifuging 2400 mL of the suspension culture and then...

Assay of Human Adenoviruses

Assay of the infectivity of human adenoviruses is an essential procedure in most experimental work on virus-host cell systems as well as for the increasing use of recombinant adenovirus vectors in clinical gene therapy protocols. Methods used for quantitative assay of adenovirus infectivity have relied largely on the plaque assay 1,2 which provides a reliable bioassay for most, but not all, of the adenovirus serotypes. The requirement for the adenovirus plaque assay is a cell type that will...

Elizabeth Perron M Behzad Zafar Amanda Pister Zhen Guo Wang Jun Tian and Prem Seth

This chapter describes several methods for recognizing apoptosis in tumor cells following infection with a replication-deficient adenovirus expressing the tumor suppressor gene p53. We include cytotoxicity assays and assays of apoptosis, including DNA-nucleosomal DNA fragmentation DNA laddering , TUNEL, DAPI staining, analysis of the sub-Gj subdiploid population, and degradation of poly ADP-ribose polymerase as assayed by Western blot . Although this is not a comprehensive list of protocols to...

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 and

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...

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

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

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

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

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...

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 William S M Wold

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

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...