ad5 genomic dna  (ATCC)

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: Segments of the Ad5 genome were individually amplified and cloned into high-copy plasmids. Each of the five central “building blocks” contains unique sequences at either end (represented by distinct colors) that create a 15–20 bp overlap with the respective neighboring block. Each block is flanked by recognition sites for the restriction enzyme BstBI. Following digestion, the unpurified plasmid fragments are directly added to an isothermal assembly reaction and then delivered to packaging cells via electroporation. This system thus facilitates the generation of infectious adenovirus particles from plasmids in three steps that can be carried out in under two hours.

Article Snippet: Overlapping regions of Ad5 genomic DNA were amplified with Platinum SuperFi DNA polymerase (Thermo Fisher Scientific).

Techniques: Amplification, Clone Assay, Blocking Assay, Plasmid Preparation, Electroporation

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: The cloned blocks 1–7 are aligned to a map of the Ad5 genome. Protein-coding genes are indicated in red, with arrows indicating the strand that is transcribed. The inverted terminal repeats (ITRs), which define the left and right ends of the adenovirus episome, and virus-associated RNAs (VA RNA) are shown. The early and late transcriptional units are marked by green and black arrows, respectively.

Article Snippet: Overlapping regions of Ad5 genomic DNA were amplified with Platinum SuperFi DNA polymerase (Thermo Fisher Scientific).

Techniques: Clone Assay, Virus

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: A. Equimolar amounts of each of the seven Adenobuilder plasmids were digested with BstBI and then incubated for an additional 60 min with (+) and without (-) the assembly enzyme mix. Aliquots containing 660 ng total DNA were assessed on a 0.8% agarose gel. Size markers (in kb) are shown on the left; the bands corresponding to the vector backbone and each of the excised blocks is indicated at right. B. Unassembled (upper panel) or assembled (lower panel) blocks were delivered to HEK293 cells by electroporation. Images were captured at 36 h, at 20X magnification. Arrows indicate representative rounded cells. Scale bar = 200 μm. C. HEK293 cells were infected with 10 μl of the primary lysate, derived directly from cells that had been electroporated (upper panel), or with 1 μl of lysate obtained after a single round of amplification (lower panel). Viral titers were determined by detection of the Ad5 hexon protein (green), which is expressed late in the viral life cycle. Magnification, 10X. Scale bar = 400 μm. D. Genomic DNAs from the reference Ad5 stock (Ref) and assembled Ad5 (AB) were compared by restriction digest. One μg of DNA from each sample was digested with EcoRV or HindIII. Fragments of the indicated sizes were resolved on 0.8% agarose. E. A viral particle in a primary, unpurified lysate was visualized by electron microscopy after negative staining, Magnification, 135000X. Scale bar = 100 nm.

Article Snippet: Overlapping regions of Ad5 genomic DNA were amplified with Platinum SuperFi DNA polymerase (Thermo Fisher Scientific).

Techniques: Incubation, Agarose Gel Electrophoresis, Plasmid Preparation, Electroporation, Infection, Derivative Assay, Amplification, Electron Microscopy, Negative Staining

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

Article Snippet: Overlapping regions of Ad5 genomic DNA were amplified with Platinum SuperFi DNA polymerase (Thermo Fisher Scientific).

Techniques: Mutagenesis, Construct, Amplification, Blocking Assay, Derivative Assay, Plasmid Preparation, Modification, FLAG-tag, Infection, Virus, Generated, Western Blot, Control

Journal: Molecular Therapy

Article Title: Generation of a Kupffer Cell-evading Adenovirus for Systemic and Liver-directed Gene Transfer

doi: 10.1038/mt.2011.71

Figure Lengend Snippet: Ad5/6GL is taken up into macrophages less than unmodified Ad5GL. (a) adenovirus (Ad) vectors were added to RAW 267.4 cells (multiplicity of infection 10,000). Cells were harvested 24 hours later and genomic DNA was extracted. Real-time PCR was used to quantify viral and cellular genomes using cytomegalovirus (CMV) and GAPDH specific primers, respectively. Data are normalized to cellular genomes. N = 4. BALB/c mice were predosed intravenous (i.v.) with 3 × 1010 virus particles (vp) in 100 µl in phosphate-buffered saline (PBS). After 4 hours, the mice were injected with 1 × 1010 vp in PBS of unmodified or chimeric Ad vector. Mice were imaged and luciferase expression was quantified (b) 24 hours after Ad vector injection and (c) over time. N = 5. (d) Varying doses of Ad vectors were injected i.v. into mice. After 1 hour, mice were euthanized and 20 ng of tissue from the large liver lobe was harvested. Total DNA was extracted and real-time PCR was used to quantify viral genomes using CMV primers. *P < 0.05; **P < 0.005; ns = not significant. Means ± SEM.

Article Snippet: Ad5/6GL: Ad6 viral genomic DNA was purified using a PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA) and the HVR cassette was amplified by PCR (F primer 5′-TACTTTGACATCCGCGGCGTGCTGG-3′ and R primer 5′-GTGGGGCCATGGGGAAGAAGGTGGCG-3′), cut with Apa I and Pst I and cloned into the Ad5GL backbone.

Techniques: Infection, Real-time Polymerase Chain Reaction, Virus, Saline, Injection, Plasmid Preparation, Luciferase, Expressing

Journal: Molecular Therapy

Article Title: Generation of a Kupffer Cell-evading Adenovirus for Systemic and Liver-directed Gene Transfer

doi: 10.1038/mt.2011.71

Figure Lengend Snippet: Expression of hFIX from intravenous (i.v.) injection of helper dependent adenovirus (Ad) vectors. Helper dependent (HD) vectors were injected intravenously into BALB/c mice at 2.5 × 1010 virus particles (vp)/mouse. Serum was harvested on (a) day 7 and (b) up to day 21. Serum was analyzed for human Factor IX by enzyme linked imunosorbent assay. N = 5 for Ad5 and Ad5/6; N = 4 for Ad6. **P < 0.005; ns = not significant. Means ± SEM.

Article Snippet: Ad5/6GL: Ad6 viral genomic DNA was purified using a PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA) and the HVR cassette was amplified by PCR (F primer 5′-TACTTTGACATCCGCGGCGTGCTGG-3′ and R primer 5′-GTGGGGCCATGGGGAAGAAGGTGGCG-3′), cut with Apa I and Pst I and cloned into the Ad5GL backbone.

Techniques: Expressing, Injection, Virus