Biomedical Engineering Reference
In-Depth Information
Many factors have been found to influence the size and charge of vector-DNA com-
plexes. Factors include buffer composition (presence of salts), nature of the cationic vector,
DNA concentration and time. These can easily be investigated using the DLS technique
[77, 97, 105].
To date, by far the most visually informative data has come from transmission electron
microscopy of the particles within transfected cells (unpublished Collins and Fabre) [106,
107]. Pre-coating of the formed particles with gold beads using a biotin-streptavidin link
where possible ensures easy and unambiguous identification following cell entry. Figure 11.4d
shows two peptide-DNA particles entering a cell by macropinocytosis; the inner particle is
enclosed within a vesicle. The particles are clearly labeled by a halo of gold beads. Electron
microscopy gives us not only information regarding shape and size of the complexes, but
also reveals valuable information about the movement of the particle through the cell.
Other ultrastructural techniques, such as atomic force microscopy and scanning electron
microscopy, have shown a variety of different shapes of vector-DNA complexes [83, 108].
However, one has to be wary of the accuracy of these findings, since preparation of the
samples for these procedures is highly unphysiological, including drying and/or high salt
concentrations, all of which greatly influence the particle structure.
11.2.6 Optimizing
in vitro
gene delivery
Prior to physical characterization of a new non-viral vector system, work must begin on
establishing its ability to delivery DNA
in vitro
. Choose a familiar cell line that is easy to
grow and manipulate, and if possible relevant to your intended end target tissue.
Outlined below are the protocols for preparation of DNA/Lipid complexes (see Pro-
tocol 11.5), preparation of DNA/peptide complexes (see Protocol 11.6) followed by the
protocol for a 'typical' transfection (see Protocol 11.7). Vector/DNA preparation and sug-
gested incubation times do not usually vary greatly between the different chemical vectors
and as explained, it is vital that some optimisation is carried out to ensure that the vector
is used at its full potential. For commercially bought non-viral vectors it is however best
to begin by following the manufacturer's guidelines but nevertheless, optimisation is still
important to achieve the highest efficiency for your application.
PROTOCOL 11.5 Preparation of Lipid-DNA Complexes
Equipment and reagents
•
Lipofectamine 2000 at 1 mg/ml (Invitrogen)
Endotoxin-free plasmid DNA at 1 mg/ml in water
h
•
Culture medium without supplements.
i
•
Method
1Dilute15
μ
l DNA in 135
μ
l culture medium (without supplements).
j
2 In a second tube, dilute 30
μ
l Lipofectamine 2000 in 120
μ
l culture medium (without
supplements).
