Biology Reference
In-Depth Information
3. Protocol 3: Preparation and Transfection of Organotypic Brain Slices with Recombinant
Adenovirus-5 Vector Containing the GFP-Apoaequorin Gene
Recombinant viral vectors, such as human adenovirus serotype 5 (Ad5), adeno-
associated virus (AAV), Sindbis viruses, or retroviruses (e.g., Lentiviruses), are
highly e
Y
cient expression vectors for gene delivery in mammalian cells or tissues
(
Tenenbaum et al., 2004
). Both Ad5 and the Sindbis virus have been used to
mediate high levels of expression of GFP-apoaequorin in primary neuronal cul-
tures, brain slices, and retinal explant cultures (
Rogers et al., 2005
). Ad5 was found
to preferentially infect glial cells in cortical- or hippocampal-derived tissue as well
as M¨ ller cells (a type of glial cell) in retinal explant cultures (
Rogers et al.,2005
).
a. Materials
Vibratome (e.g., Model VT-1000, Leica)
Gas tank (95% O
2
/5%CO
2
)
Glass petri dish (3-5 cm diameter)
Membrane filter inserts (e.g., 12 mm Transwell
Ò
Permeable Supports, Corning)
12-well tissue culture plates
Artificial cerebrospinal fluid (ACSF), pH 7.4 (124 mM NaCl, 3 mM KCl, 2 mM
CaCl
2
, 1.3 mMMgCl
2
, 25 mMNaHCO
3
, 1.25 mMNaHPO
4
, and 10 mM glucose)
Superglue, Blades, Low temperature melting Agar, Culture medium (50 %
MEM, 25 % HBSS, 25 % Horse Serum, 6.5 mg/ml glucose, 2 mM L-glutamine,
100 U/ml penicillin, 100
m
g/ml streptomycin, pH 7.2).
Ad5-GA Viral particles (1
5x10
8
particles).
Specimen chamber for live imaging
b. Methods
Organotypic slices can be prepared similarly to previously reported methods
(
Stoppini et al., 1991
). Briefly, prepare 400-600
m
m slices as described in section 3b.
Once the slices are cut, gently transfer them using a Pasteur pipette onto a culture
membrane insert and into a 12-well culture plate containing prewarmed culture
medium. Slices can be kept in culture for 4-5 days and viral particles added directly
to the medium approximately 48 h prior to imaging. After viral transfection and
verification of GFP expression, the membrane culture insert with attached slice can
be moved to a larger Petri dish containing growth media (e.g., 35 mm) and the
membrane excised with a scalpel blade. Care should be taken to avoid folding of
the membrane insert, which will cause injury to the tissue. The membrane with
attached slice should then be carefully inserted into an imaging chamber and a
tissue anchor placed on top to secure the tissue (e.g., Series RC-20, Harvard
Apparatus). Once the slice is mounted onto an inverted microscope, a simple
gravity flow system for delivering bu
er together with a small peristaltic pump
connected to the output can be used for perfusion (
Mohammed et al., 2007
).
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