Protocol for

Adeno-1 Expression System Manual

Link to Full Protocol: http://www.capitalbiosciences.com/system/data/files/Adeno1_M ...

Abstract or Description:

The Adeno-1™ Expression System provides an efficient method
for constructing recombinant adenoviruses. Our procedure uses in
vitro ligation to subclone your gene of interest into a replication!
incompetent (-E1/-E3) human adenoviral type 5 (Ad5) genome.
This approach enables you to produce recombinant adenoviruses
quickly (less than 3 weeks) and reliably. Our pShuttle vectors (+/-)
have very flexible cloning sites, which allow easy subcloning
manipulation.

Cloning your gene of interest into the pShuttle (+/-):
1. Subclone your gene of interest into the pShuttle(+/-) using any of
the unique restriction sites located in the MCS region.

2. PI-Sce I/I-Ceu I digestion of recombinant pshuttle and adeno DNA

a. Prepare a 30ul PI-Sce I/I-Ceu I double-digest of your pShuttle
and adeno DNA.

b. Combine the reagents shown in Table 2 in a sterile 1.5 ml

microcentrifuge tube.

c. Mix well and spin briefly to collect liquid.
d. Incubate at 37ºC for exactly 3 hours.

e. Perform phenol:chloroform:isoamyl alcohol (25:24:1)
extraction by using the protocol in Appendix A.

Table 2. PI-Sce I/I-Ceu I Double-Digest of pShuttle and pAdeno DNA

Reagent Volume
pShuttle Plasmid or pAdeno DNA 2-3ug

10X Double Digestion Buffer 3ul
PI-Sce I Restriction Enzyme (1 unit/ul) 2ul

I-Ceu I Restriction Enzyme (1 unit/ul) 2ul
10X BSA 3ul

Sterile H2O x ul
Total Volume 30ul

Produce recombinant adenoviral DNA:
1. Subclone your expression cassette into the Adeno genome:

a. Combine the reagents shown in Table 3 in a sterile 1.5 ml
microcentrifuge tube in the order as shown.

b. Gently mix, then spin briefly in the microcentrifuge.
c. Incubate at room temperature for 1-2 hours.

d. To the ligation product, add the following:

- 1.5 ul 10X Swa I digestion Buffer

- 1.5 ul 10X BSA
- 1.0 ul Swa I enzyme

- 1.0 ul ddH2O

e. Incubate at room temperature for 1-2 hours.

f. Perform phenol:chloroform:isoamyl alcohol (25:24:1)
extraction by using the protocol in Appendix A.

Table 3. Ligation of pShuttle to Adenovector
Reagent Volume

PI-Sce I/I-Ceu I digested vector 4ul
PI-Sce I/I-Ceu I digested pShuttle 3ul

5X DNA Ligation Buffer 2ul
DNA Ligase 1ul

Total Volume 10ul

2. Transform E.coli with ligation products

a. Transform electro! or chemically competent DH5alpha or our Ad-
competent (Cat No. DA014) cells with the Swa I digestion product.

b. Select for ampicillin-resistant (Ampr) transformants by plating
the transformation mixture on a LB agar/Amp plate (100 ug/ml ampicillin). Incubate overnight at 37ºC.

c. Pick up 10 colonies and inoculate into 100 ul LB/Amp medium
in a sterile Eppendorf tube in the early morning.

d. After 5-6 hours of incubation, screen for positive clones using
PCR with Adeno screening primer mix (using the protocol in

Appendix B).
e. When you have identified a bacterial clone carrying the

desired recombinant, inoculate 100-500 ml of LB/Amp
medium with all the bacteria (100 ul) from the 1.5 ml

Eppendorf tube.
f. Incubate the culture at 37ºC overnight.

g. Purify the plasmid using our Column Pure Plasmid Maxi-Prep
Kit (Cat No DD205) or the Qiagen kits. Expected yield: 30-50 ug

plasmid DNA/100 ml culture.
Note: pAdeno is a large plasmid (>30 kb) that is susceptible to damage and

rearrangement in E.coli. For best results, always use fresh, log!phase
cultures for purification of recombinant pAdeno DNA. Do not store your

culture at room temperature, 4ºC, or on ice for long periods ( i.e. >24 hours)
before starting purification.

3. Analyze putative recombinant Adenoviral DNA by PCR or
restriction digestion

PI-Sce I and I-Ceu I Restriction Analysis:

a. Set up 30ul PI-Sce I/I-Ceu I double digests by combining the
reagents in Table 4 in a sterile 1.5 ml microcentrifuge tube.

b. Mix well and spin briefly to collect contents.
c. Incubate at 37ºC for exactly 3 hours.

d. Verify the insert by electrophoresis on a 0.8-1%
agarose/SafeView™ (Cat.No DG108) gel.

Important: Since I-Ceu I and PI-Sce I tend to remain bound to DNA, use a
gel loading buffer that contains SDS (final concentration after combining

with sample: 0.1%), heat to 65ºC for 10 minutes before loading.

Table 4. Restriction Analysis of Recombinant Adenoviral DNA

Reagent Volume
Adenoviral DNA 3ug

10X Digestion Buffer 3ul
10X BSA 3ul

PI-Sce I (1 unit/ul) 2ul
I-Ceu I (1 unit/ul) 2ul

ddH2O x ul
Total Volume 30ul

PCR Analysis:

You can also screen pAdeno DNA for the presence of pShuttle
derived expression cassettes by using PCR with Adeno forward

and reverse PCR primers (using the protocol in Appendix B).
These primers specifically amplify a 350-bp sequence that spans

the cloning site in pAdeno. Only recombinant pAdenoviral
templates are amplified since non!recombinants lack the pShuttle

sequence needed for annealing with the reverse primer.

4. Preparing Recombinant Adenoviral DNA for Transfection

a. In a sterile 1.5 ml microcentrifuge tube, combine the reagents
in Table 5.

b. Mix contents and spin the tube briefly.
c. Incubate at 37ºC for 2 hours

d. Perform phenol extractions by using the protocol in
Appendix A.

Table 5. Pac I Digestion of Recombinant Adenoviral DNA

Reagent Volume
Recombinant Adenoviral DNA 3ug

10X Pac I Digestion Buffer 4ul
10X BSA 4ul

Pac I (10 units/ul) 2ul
ddH2O x ul

Total Volume 40ul

Generate the recombinant adenovirus in packaging cells:

1. Plate 293 cells at 70% confluency in a 6-well plate 12-24 hrs

before transfection.

2. Incubate the plate(s) at 37ºC in a humidified atmosphere

maintained at 5% CO2.

3. Transfect each 6 well culture plate with 10 ul of Pac I-digested

recombinant Adenoviral DNA. Use Celfectin™ (Cat.No DG061)
to transfer DNA into 293 cells.

4. Subculture cells into a T75 flask using trypsin 3-5 days after

transfection.

5. Check periodically for cytopathic effect (CPE). Note: It

normally takes 7-12 days to see CPE.

6. When >90% of the cells have detached from the plate, prepare

viral stock by following steps 7-10. Name this stock “Primary
Amplification” and store at –20ºC.

- Primary Amplification Stock is suitable for infecting
target cells. We suggest you evaluate the function of this

viral stock before preparing High-Titer Stock.
- The presence of your recombinant construct can be

verified by PCR or Western blotting.

7. Centrifuge the suspension at 1,500 x g for 5 min at room

temperature.
8. Re-suspend the pellet in 500 ul sterile PBS

9. Lyse cells with three consecutive freeze-thaw-cycles. Freeze

cells in dry ice/ethanol bath, thaw cells by placing the tube in a
37ºC water bath. Do not allow suspension to reach 37ºC.

10. After the third cycle, briefly centrifuge to pellet debris.

Transfer the lysate to a clean, sterile centrifuge tube and store
the lysate at -20ºC.

11. Determine the adenoviral titer. The Adenovector™ Rapid Titer

Kit (Cat.No DA1-20T) enables you to determine the adenoviral
titer by using an anti-Hexon antibody cell staining assay.

Infecting target cells:
If the viruses are to be used in in vitro cell cultures, Double CsCl
purification is not required as the viral supernatant will provide
100% gene transduction efficiency in most human cell lines. For in
vivo studies (i.e. animal studies), purification is essential to remove
defective particles, cell debris, and small amounts of media
components, since these contaminants can induce significant
immune responses. In addition, CsCl purification will concentrate
the virus to a level suitable for in vivo injections.

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Posted By: kevin on 11/24/2009 9:58:55 AM

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