Infection with retroviruses
It is well established with avian retroviruses that cells
are most efficiently infected just after they are trypsinized.
Trypsinization does two things. First, it apparently exposes
the receptor to which the virus binds. Additionally, it stimulates
DNA synthesis and cell division, two processes that are essential
for the establishment of retroviral infection.
Additionally, it is important to do the infection in as small
a volume of medium as possible.
It is likely that these principles are also relevant to the infection
of mammalian cells with murine retroviruses.
Infection with retroviruses is facilitated
greatly by polybrene. This is a small, positively charged
molecule that binds to cell surfaces and neutralizes surface
charge. This apparently allows the viral glycoproteins to bind
more efficiently to their receptors, because it reduces the repulsion
between sialic acid-containing molecules. To use polybrene optimally,
the cells to be infected should be pre-treated with polybrene
and the virus adsorption should be done in the presence of polybrene.
Cells vary in the amount of polybrene that they will tolerate.
From 1 to 10 µg per ml is usual. Some of these values can
be found in the Cell Line HyperCard stack.
There are two ways to infect adherent cells.
What I like is to seed the cells to be infected in medium containing
polybrene and wait until the cells attach to the dish. This can
take from 1 to 4 hrs. Then remove the medium, add the virus,
let it adsorb for 30 to 60 min and then add back medium containing
Alternatively, you can suspend trypsinized cells in a small volume
of medium containing the virus and let adsorption occur while
the cell suspension is incubated at 37 °C. The infected cells
can then be seeded on dishes.
Retroviral infection requires cellular DNA synthesis--because
viral DNA synthesis requires many of the same components and
enzymes as cellular DNA synthesis, and mitosis--because the viral
DNA must gain access to chromosomal DNA and cannot enter interphase
nuclei. What is usually done is to infect cells at a density
such that they will grow for about 3 days before reaching confluence.
The plating efficiency of mammalian cell lines is usually high,
close to 100%. The doubling time is approximately 18 to 24 hr.
Therefore, if you seed 1 x 105 cells on a 50 mm dish, you will
probably have 2 x 105 24 hours later. Confluence is at approximately
106 3T3 cells per 50 mm dish.
Infecting lymphoid cells.
Non-adherent cells can be pelleted and then resuspended in medium
containing a retrovirus and an appropriate concentration of polybrene
and incubated with the virus for 30 to 60 min. The cells can
then be diluted with medium and allowed to grow.
We have had good success recently infecting lymphoid cells by
co-cultivation of them with virus-producing cells for 18 hours
in medium containing polybrene. In the case of COS cells, the
transfected COS cells don't survive long enough to contaminate
the lymphoid cells.
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