Biology Reference
In-Depth Information
This laser-based nanoparticle tracking system is allows nanoscale
particles such as viruses and virus aggregates to be directly and
individually visualized in liquids in real-time, which allows a high-
resolution determination of particle size and distribution. The
technique is rapid and accurate (we note highly reproducible par-
ticle titers when directly compared to microBCA determination of
protein levels). The technology measures simultaneously and
directly measuring the diffusion coeffi cient of individual particles,
using Nanoparticle Tracking Analysis (NTA) to automatically
count and size the viruses and aggregates in a sample.
The NanoSight™ system works optimally within the range of
10
6
to 10
10
viral particles per ml, and is ideally suited to nanopar-
ticles 30-1,500 nm in size, and thus is ideal for detection of adeno-
virus (~90 nm). The system would not be suitable, however, for
AAV preps, due to their smaller size. The tracking technology is
based on speed of movement of nanoparticles under Brownian
motion since particles of different sizes will scatter light differently.
We have found that in addition to basic QC checks, the system can
also be useful for studying interactions of adenoviral particles with
human proteins in solution, especially to study whether such inter-
actions can result in particle aggregation. The system can also be
purchased with fl uorescence fi lters in order detect and quantify
fl uorescently labelled particles.
3
Methods
3.1 Generation
of Ad5 Vectors with
Mutated Penton
and Hexon Proteins
To facilitate cloning strategies, we design and use shuttle plasmids,
typically 4-8 Kb in size encoding only a fragment of the Ad genome
that are (due to their reduced size relative to the Ad genome) more
amenable to manipulation of individual genes within the Ad5
genome. We aim to construct shuttle plasmids that contain the gene
for manipulation and a minimum of 500 bp (ideally 1-3 Kb) of
DNA sequence fl anking this region to facilitate homologous recom-
bination. For such strategies, it is critical to identify a unique restric-
tion endonuclease site in pAdEasy-1, which is present in the target
viral gene. For penton base,
Pme
I is unique in the Ad5 genome and
therefore linearizes pAdEasy-1 in the penton base gene to allow
homologous recombination within this specifi c region.
3.1.1 Generation of an
Ad5 Penton Base Shuttle
Plasmid
1. First use oligonucleotides 1 and 2 to PCR amplify a 4.6 Kb
fragment from the AdEasy genome containing the entire pen-
ton sequence, fl anked by 1.9 Kb (left) and 1 Kb (right) regions
of homology from the Ad5 genome.
2. Confi rm the correct size of the generated DNA fragment via
agarose gel electrophoresis.
