Biology Reference
In-Depth Information
3.3.5 Decision Point:
Intermediate Quality
Control
1. Digest the genomic DNA obtained in step 5 from procedure
in Subheadings 3.3.3 and 3.3.4 with an appropriate restriction
enzyme ( see Note 12 ). In parallel, digest 100-200 ng of the
linearized HC-Ad vector plasmid obtained in step 3 of the
rescue protocol and 100-200 ng of linearized helper virus
plasmid and analyze all samples by agarose gel electrophoresis/
ethidium bromide staining. In the case of successful HC-Ad
vector amplifi cation there should be prominent bands visible in
the lane loaded with genomic DNA from the fourth amplifi ca-
tion step, which correspond to the HC-Ad vector plasmid con-
trol. The intensity of these bands should be equal or higher
compared to the intensity of the bands which correspond to
helper virus genomes. If the intensities of the HC-Ad vector
bands is signifi cantly weaker than the intensity of the bands
corresponding to helper virus genomes repeat procedure in
Subheading 3.3.4 until prominent HC-Ad vector bands
become visible in the gel ( see Note 13 ). If prominent HC-Ad
vector bands are visible proceed with the medium-scale prepa-
ration of HC-Ad.
The goal of a medium-scale preparation of the HC-Ad vector at
this stage is to obtain the vector in highly purifi ed form and in
quantities which are suffi cient to perform small-scale functional
analysis and, importantly, allow to infect a large number of producer
cells in suspension culture in a defi ned manner to obtain large
quantities of the HC-Ad vector. A typical yield of a purifi ed HC-Ad
vector from a medium-scale preparation as described in the
following is 1E11-1E12 vector particles.
3.3.6 Medium-Scale
Preparation of HC-Ad
1. Release vector particles from the cells obtained in procedure in
Subheading 3.3.4 by three repeated freeze-thaw cycles in liq-
uid nitrogen and a water bath (37 °C). Gently swirl the tubes
with the cell suspensions while freezing and thawing.
2. Replace the medium of twelve 150-mm dish with 90 % confl u-
ent 116 cells seeded 2 days before with 15 mL of fresh, supple-
mented medium.
3. Infect cells with 0.3 mL of the freeze-thaw lysate per dish as
obtained in step 1 . Coinfect the cells with 15-20 MOI of
helper virus. One hour after infection fi ll up to 30 mL with
supplemented medium per dish.
4. 48 h after infection the cells should show a complete cyto-
pathic effect (CPE) characterized by cell rounding and detach-
ment from the dish. Harvest the cells in their medium by
pipetting up and down or scraping.
5. Spin down cells at 400 × g for 10 min. Aspirate and discard
supernatant. Resuspend the cells in 4 mL PBS. Snap-freeze the
cells in liquid nitrogen and store prior to further use at −80 °C
or immediately proceed with purifi cation.
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