Transfection of Viral DNA

The cell lines 293 and W162 are grown as monolayers in DMEM supplemented with 10 FCS, 100 U of penicillin, and 100 g of streptomycin per mL in a 7 CO2 atmosphere at 37 C (see Note 4). 2. For transfection of viral DNA, grow cells to 50-70 confluency in 60-mm tissue culture dishes. Use cells at a low passage number. 3. Digest the recombinant bacmid DNA with PacI to remove the bacterial vector (pPG-S2) from the Ad5 genome. 4. Precipitate the DNA with isopropanol and wash with 70 ethanol. 5....

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...

Virus Harvesting and Concentration

Negative Staining Electron Microscopy

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...

Construction of Full Length Plasmids by Homologous Recombination in E coli

Isolate a KpnI-SwaI DNA fragment from plasmid pBAV-301.G5FK (Fig. 1B) or PacI-NotI fragment of plasmid pBAVNotYFP (Fig. 2) using QIAquick Gel Extraction kit. 2. Mix the KpnI-SwaI DNA fragment with Sr I-digested plasmid pFBAV-302 (E3-deleted BAdV-3 DNA (13,22)) or the PacI-NotI fragment with BsaBI-PmeI digested plasmid pFBAV3 (20) (see Note 3). Add the individual mixtures into 100 L of chemically competent E. coli BJ5183 and incubate on ice for 30 min. Heat-shock the cells for 1 min at 42 C...

Construction of Transfer Plasmids

Genomic Dna Clone Diagram

Construction of Transfer Plasmid Containing Chimeric Fiber by Overlap Extension PCR Although we are describing here the construction of chimeric fiber in which the knob region of BAdV-3 fiber has been replaced with the knob region of HAdV-5 fiber (11) (Fig. 1A), the method used for creating chimeric fiber should be applicable to replacement of the BAdV-3 knob with any other adenovirus fiber knob. 1. Purify plasmid DNA containing the gene encoding BAdV-3 (5) or HAdV-5 fiber (24) using...

Temperature Sensitive Replication Competent Adenovirus shRNA Vectors to Study Cellular Genes in Virus Induced Apoptosis

Thirugnana Subramanian and Govindaswamy Chinnadurai Summary The use of shRNA for knockdown of gene expression is a powerful method. In addition to transient transfection of RNA oligonucleotides, various DNA-based vectors that express short hairpin RNAs have been successfully used for efficient depletion of gene products. Replication-defective retrovirus and adenovirus (Ad) vectors have also gained wide usage. The extension of shRNA technology to replication-competent Ad would be desirable to...

General Care and Handling of Syrian Hamsters

Housing and General Care of Hamsters 1. Hamsters should be housed two to three animals per cage (see Note 4) in polycarbonate mouse hamster cages with rodent bedding placed in the bottom of the cage for nesting. Use a stainless steel wire bar lid that snaps down tightly to prevent hamsters from escaping. 2. Use relatively slow and deliberate movements when handling hamsters. Hamsters are relatively easy to handle, but may be momentarily aggressive when suddenly startled or awakened by a...

Materials

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....

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...

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...

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....

Quantitation of Virus by Titration

Trypsinize a monolayer of MDBK cells in a T150 flask. 2. Add 100 L of cell suspension (5 x 105 cells mL in MEM plus 10 FBS) in each well of a 96-well plate. 3. Place the plate into an incubator. Meanwhile, thaw an aliquot of virus stock. 4. Aliquot 1.8 mL of serum-free MEM into each of 10 small glass test tubes with caps and number and order these tubes from 1 to 10. 5. Add 0.2 mL of virus to tube 1. Vortex and remove 0.2 mL from tube 1 and deposit it into tube 2. Vortex and repeat for the...