Biology Reference
In-Depth Information
Remove neutral red and place on a white transilluminator to
capture images (e.g., GelDoc imaging system) ( see Note 5 ).
3.4 Antitumor
Effi cacy In Vivo
1. To implant tumors prepare a suspension of exponentially grow-
ing cells (subconfl uent plates). The cells implanted to generate
tumors vary from 10 6 to 10 7 according to every cell line.
2. Inject 0.15 mL of cell suspension in each fl ank of the animal
(mouse or hamster) with a 29 G needle.
3. Follow up animals every 3 days and measure length ( A ) and
width ( B ) of each tumor with a caliper. Volume
(mm 3 ) = A × B × (
/6). Once the tumors reach 100-150 mm 3 ,
animals must be distributed randomly in groups.
4. Dilute purifi ed adenovirus in PBS for injection. For intrave-
nous or intraperitoneal administration use a volume of 200
π
L
(typically we use 5 × 10 10 or 10 11 vp in a 25 g mouse and 4 × 10 11
in a 100 g hamster). Inject in the lateral tail veins rubbing
them with alcohol for dilation, using a 1 mL syringe with a
30 G needle. For intratumoral injection used anesthesia, inject
a maximum of 20
μ
L/tumor using small syringes (0.3 mL)
and 30 G needles, and distribute this volume is multiple (2-4)
injections. Wait for 1 min before removing the needle and
between injections. To avoid the leakage of the injected virus
upon sequential intratumoral injections it is recommended to
seal the injection puncture with tissue adhesive.
5. Follow tumor volume with a caliper every 2 or 3 days. Animals
must be handled according animal use and care committee rec-
ommendations, usually avoiding ulceration and size over 1 mL.
μ
3.5 Adenovirus
Genome
Biodistribution by
qPCR
1. Sacrifi ce animals (CO 2 chamber) and resect organ (tumor or
others) into eppendorf tubes to snap freeze in liquid nitrogen
or in dry ice/ethanol. Homogenize the tissue in liquid nitro-
gen with a pestle and mortar on dry ice until a powder is
obtained. Extract total DNA using commercial kits (e.g.,
NucleoSpin DNA Blood, Macherey-Nagel; QiAamp DNA
Mini, Qiagen).
2. Aliquots of resuspended homogenate before lysis from a nega-
tive tumor or tissue (vehicle control animals) can be spiked
with nine serial dilutions of purifi ed virus of known concentra-
tion (from 10 to 10 9 vp/
L) to generate a standard curve.
3. Dilute DNAs to a fi nal concentration of 25 ng/mL.
4. Mix 4
μ
μ
L of each DNA sample (100 ng), with 0.3
μ
L of 10
μ
M
hexon primers, 0.1
μ
L probe, 5
μ
L of 2× Premier Extaq, and
L of H 2 O.
5. Amplify in LightCycler (Roche) heating at 10 min a 95 °C fol-
lowed by 40 cycles of 95 °C—15 s/60 °C—1 min, to obtain
the amplifi cation curves of the standard curve and samples.
0.3
μ
Search WWH ::




Custom Search