Biomedical Engineering Reference
In-Depth Information
11. Purifi cation (optional).
(a) Centrifuge (SW41 rotor) the media containing virus at
23,500 RPM for 2 h.
(b) Immediately pour off the media leaving an invisible pellet.
Add 60
l of serum-free media to the invisible pellet, and
keep at room temperature for a few minutes.
(c) Pipette the concentrated virus from each tube and combine.
(d) Make 10
ʼ
l aliquots in Eppendorf tubes.
12. Verify virus production using Lenti-X GoStix™ (
see
protocol
PT5123-2 for details) or titrate the virus stock, and then use the
virus to transduce target cells or store at −80 °C (
see
Note 4
).
ʼ
1. Prior to performing any procedures involving animals, the
operator should ensure that the surgical area is clean and well
organized and contains all required instruments. Tools can be
sterilized by placing them in a fl ash bead sterilizer for a short
time. This sterilization technique is suffi cient for rodent sur-
gery but not for larger animals.
2. Lay out all the surgical instruments on an absorbent pad in the
order in which they will be used. Inspect the stereotactic frame
to ensure that it is in a proper operating condition. Ensure that
all the manipulator arms move freely and smoothly with little
to no sideways movement in the syringe holder. Direct the
light beam at the ear bars and position the microscope such
that the surgical area is visible through the eyepiece.
3. Place the adult rat (7-10 weeks old) under anesthesia by intra-
peritoneal (IP) injection of ketamine (75 mg/kg) and dexme-
detomidine (0.25 mg/kg). Before commencing surgery,
administer carprofen subcutaneously (5 mg/kg) as an analge-
sic, and ensure that the rat is completely sedated by pinching
the footpad or tail.
4. Using surgical shavers, remove enough fur from the head of
the rat to allow for aseptic procedures.
5. Carefully mount the rat onto the stereotactic frame and use the
incisor bar to loosely immobilize the skull. Raise the rat to the
level of the ear bars. Proceed by supporting the head of the rat,
and slide one of the ear bars into the ear canal and tighten in
place. While keeping the head supported, slide the other ear
bar into the proper position. The ear bars are meant to prevent
any mediolateral head movement while maintaining the dorso-
ventral axis rotation free (
see
Notes 5
-
7
).
6. Use blunt forceps to open the mouth of the rat, and insert the
incisor bar. Press down gently to ensure that the incisors are well
seated. Swing the nose clamp over the nose of the rat and tighten
gently. Ensure that there is no disruption in respiration.
3.2 Generation
of Glioma Models:
Intracranial Lentiviral
Injection in Rats
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