Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 22

4 Notes

1. It should be noted that the GFP (or RFP) is expressed inde-
pendently to the sequence of interest. Since it is controlled by
a CMV promoter, the GFP is nonspecifi cally expressed in virally
infected cells. Thus, GFP can be used as index for viral infection.
On the other hand, the expression of interest genes, especially
those controlled by specifi c promoter, may be expressed in
selected cell types but not all of virally infected cells.

2. When designing primers for amplifi cation of the gene of inter-
est, it is suggested that (1) use cDNA and DNA from same
species of animal to be used in the study; (2) it is important
to include an RNA polymerase binding site, such as a Kozak
consensus sequence (ACC AUG G or extended form:
GCCGCCACCAUGG) at 5 of coding region. Here, AUG is

starting codon (ATG for DNA). Without an RNA polymerase
binding site, the translation may not be processed. In some
cases, even with Kozak sequence, the gene may still not be
expressed. We found that including 5

′

noncodon region may
facilitate gene expression. (3) It is recommended to include
restriction enzyme sites in primers, so that it can be digested by
restriction enzymes for insertion into shuttle vectors.
3. Do not regrow BJ5183 cells containing recombinant adenovi-
rus plasmid because of the recombination and rearrangement
features of BJ 5183 cells.
4. In order to obtain high-titer virus, it is critical that infected
cells reach the desired CPE (i.e., all of cells become round and
50 % of the cells are detached) in 3-5 days. Shorter or longer
times required to reach the desired CPE reduce the yield of
recombinant adenovirus, resulting in a faint band or no
detected band is after CsCl gradient centrifugation.
5. In contrast with pAdTrack (SES-HUS) vectors in which GFP
(or RFP) can be used to monitor the transfection and infec-
tion, pShuttle vectors cannot be monitored except with the
CPE. Therefore, it is critical to verify the insert in the viral
lysate after transfection and infection.
6. Dialyzed recombinant adenovirus is not stable at room tem-
perature. It can be inactivated in 30 min at room temperature,
whereas it can stay stable in ice for 3-5 h. Thus, it is essential
to keep the virus in ice until injection. Also, if the injection
lasts for more than 10 min, it is recommended to take only
5-10

′

l of the viral solution at a time and refi ll the solution for
each injection.
7. The coordinates of stereotaxic injection are based on brain
atlases such as for mice or rats [ 16 , 17 ]. However, the effects

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