Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 18

Recombination of shuttle vector into AdEasy-1 vector. The shuttle
vector containing the sequence of the gene of interest is linearized
by digestion with Pme I (or Eco RI or Bst1107 I, if the insert con-
tains Pme I). The linearized shuttle vector is then transformed into
AdEasier cells, whereby the shuttle vector recombines into the
AdEasy1 vector. The recombinant adenoviral colonies are then
selected by kanamycin, since shuttle vectors are kanamycin resis-
tant, while AdEasy vectors are ampicillin resistant. The smallest
colonies should then be picked and minipreps is prepared. The
size of supercoiled plasmids is checked using a 0.7 % agarose
gel. The recombinant adenoviral plasmids run slower than 12 kb,
whereas shuttle vectors alone run about 5 kb [ 14 ]. The candidate
plasmids can be further digested by Pac I, which should yield a
large fragment (>33 kb) and a smaller fragment (3 or 4.5 kb)
[ 14 ]. The correct colonies are transformed into a strain of E. coli
cell that has high plasmid propagation but not recombination
features, such as DH10B, to generate more plasmids ( see Note 3 ).
The plasmids are further confi rmed using restriction enzyme diges-
tion, such as with Hind III, and PCR. The confi rmed plasmids are
further used for generation of high-titer recombinant adenovirus.
Generation of high-titer recombinant adenovirus: after linear-
izing with Pac I, the recombinant adenoviral plasmids are trans-
fected into HEK 293 cells as described by He et al. [ 12 - 14 ].
Briefl y, the linearized plasmids are transfected into HEK 293 cells
using a standard transfection approach such as LipofactAMINE.
Two to three weeks after the transfection, when all of cells have
become round and 50 % of the cells are detached (cytopathic effect,
CPE), the cells can be collected by scraping them with a cell scraper
(do not use trypsin). The cell lysate is then used to infection more
cells to generate high-titer recombinant adenovirus. If it is desired,
the supernatant (media) can also be used to further infect cells,
which can accelerate the production of high-titer adenovirus.
To obtain high-titer adenovirus, 15-20 75 cm fl asks of viral-
infected HEK 293 cells are needed, which may require two to four
rounds of infections starting from the viral lysates (may be media)
from transfected cells above ( see Note 4 ). The viral lysates are fur-
ther used to infect HEK 293 cells until infected HEK 293 cells are
suffi cient for preparation of high-titer adenovirus (15-20 fl asks).
High-titer adenovirus is prepared as described in previous proto-
cols [ 13 , 14 ] ( see Note 5 ).
Check titer of recombinant adenovirus: several methods can be
used to check the titer of recombinant adenovirus as described by
Luo et al. [ 14 ]. We have used GFP (or RFP) expression to check
the titer of adenovirus. HEK 293 cells are plated in 12-well plates
with 1 ml medium per well. Next day, the high-titer recombinant
adenovirus is serially diluted using tenfold dilution intervals. Ten
microliters of diluted viral solution (usually 10 3 and 10 8 dilutions)
are added into each well, respectively. The infected cells (GFP or

3.2.2 Recombination
of Shuttle Vectors into
AdEasy-1 Vectors and
Generation of High-Titer
Recombinant Adenovirus