Biomedical Engineering Reference
In-Depth Information
•
Cell death occurs rapidly after infection with virus containing cell lysate.
—Dilute the virus stock further before addition to cells; it may be of a very high titer
(see note c in Protocol 11.2).
—Ensure that virus is not contaminated with fungus or bacteria. It is possible to filter
sterilize the original cell lysate using a 45
μ
m syringe filter; however, the titer may be
significantly reduced.
11.3.1.3 Non-viral vectors
•
Poor transfection efficiency
. Try optimization strategies suggested in Section 11.2.7 to
improve transfection efficiency. Look at cytotoxicity (Section 11.2.9).
With polycationic vectors, transfection efficiency is often reduced in the presence of
serum and other proteins. Therefore, they should be avoided in the culture medium, along
with other polyanions or cations such as HEPES which have also shown to disrupt gene
delivery.
Likewise, antibiotics should be avoided in all cell culture media. Antibiotics mask
problems with contamination in the cell culture, which could affect transfection efficiency.
It has also been found to interfere with some non-viral vector systems (e.g. Lipofectamine,
Invitrogen).
•
Variable or irreproducible results
. Standardize all components of the delivery system.
Prepare a large batch (5-10mg) of endotoxin-free DNA (i.e. using the Gigaprep assay
from Qiagen), resuspending the final pellet in sterile filtered water and storing in small
aliquots at
35
◦
C. Do not refreeze DNA once thawed.
Where possible, similar concentrations of non-viral vectors should be prepared in the
appropriate sterile filtered buffers and stored in small aliquots at
−
35
◦
C. This can be
easily carried out for peptides and PEI. Lipofectamine products, however, must usually
be stored at 4
◦
C; follow manufacturer's guidelines for shelf life.
There is often variability between different experiments. It is essential to include
controls in each experiment, since precise levels of transfection cannot be effectively
compared between two separate experiments. This is due to assay sensitivity with some
reporter genes, in particular the luciferase assay, which is highly sensitive to temperature
and light. Controls should include cells alone, DNA alone, a positive control and a control
vector where possible. For some assays, internal standards can be included by aliquoting
and storing a known positive sample which can be included in each assay.
−
•
Toxicity
. If toxicity is observed, try different optimization strategies (see Section 11.2.7),
including reducing the time of cell exposure to transfection agents, reducing the concen-
tration of vector/DNA complexes and addition of serum within the culture medium.
References
1. Neschadim, A., McCart, J.A., Keating, A. and Medin, J.A. (2007) A roadmap to safe, efficient,
and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation.
Biology of
Blood and Marrow Transplantation
,
13
(12), 1407-1416.
2. Gordillo, G.M., Xia, D., Mullins, A.N.
et al
. (1999) Gene therapy in transplantation: patho-
logical consequences of unavoidable plasmid contamination with lipopolysaccharide.
Transplant
Immunology
,
7
(2), 83-94.
