& Adenovirus Cre - information & protocol
||MEM (Sigma catalog #M-0268)
||2 M CaCl2
|| *Adenovirus - University of Iowa
The University of Iowa facility sends virus at a titer of 10E12 particles/mL
which is equal to 10E10 PFU/mL. It is important to minimize freeze/thawing
as the titer drops ten-fold each time the virus is frozen. Upon arrival
we thaw the virus and aliquot it. Then we thaw an aliquot when we are
ready to use it. However, Iowa accounts for one thaw/freeze to aliquot,
so no need to adjust for this. Therefore, at the time of infection the
titer is actually 10E10 PFU/mL. Ordering info. can be obtained from the
The person dealing with the ordering is:
Senior Research Assistant, Manager
University of Iowa
Gene Transfer Vector Core
Iowa City, IA 52242
phone: (319) 335-6726
fax: (319) 353-5572
The director of the facility:
Beverly L. Davidson, Ph.D.
Roy J. Carver Assoc Prof Internal Medicine
Assoc Prof in Neurology and Physiology & Biophysics
Director, Gene Transfer Vector Core
University of Iowa College of Medicine
Iowa City, IA 52242
Formation of AdCre:CaPi co-precipitates:
We typically use 2.5 x 10E7 PFU as our standard dose. You
can change the titer by altering the amount of virus used, and adjusting
the volume of MEM accordingly in order to give a final volume of 125 uL.
When less virus is needed, we normally dilute it in MEM and then use 2.5
Add 2.5 uL of AdCre to 121.9 uL of MEM and mix well. Next
add .6 uL of CaCl2 and mix well. Let this mixture sit for ~20 minutes
prior to use.
1) Anesthetize the mice with avertin, making sure that the mice are fully
anesthetized and have no reaction to pain. I test this by pinching their
toe with a pair of forceps. If they are not fully anesthetized they will
shake their head scattering the virus.
2) Using a protein gel loading tip, take 62.5 uL (half) of the virus mixture
and place the pipet tip at the opening of one nostril. It won't fit into
the nostril, but that's fine. Slowly expel the virus solution from the
tip. Be sure the mouse is indeed inhaling the drop that is forming. If
not, stop expelling and wait until it inhales what is there.
3) Let the mouse recover for 5-10 minutes. It will cough for a while after
inhaling the virus. Wait until its breathing has returned to normal.
4) Repeat with the remaining 62.5 uL of virus mixture.
Note*- Occasionally, mice don't cope with the avertin very well. It depresses
their temperature and breathing rate. If you notice a mouse that is breathing
really slowly or shivering, filling a large glove with hot water and placing
the mouse on the glove often helps it to recover & survive.