1. Preperation
First thing you'll have to do is to
prepare the shuttle vector carrying the your gene of interest in the right
orientation, with a kozak sequence and certainly a Startcodon, all this is
not provided in the pAdTRACK CMV shuttle vector.
The following procedure is a recommendation
to achieve this plus the presence of a tag in the resulting protein. As a
start I used our standard pCDNA3 vector carrying the Myc sequence, propably
you'll have your gene in such a vector anyway, if not ligate it into this
vector using our standard XhoI/NotI cloning strategy. If you have your vector
confirmed you cut your insert out using an EcoRI/NotI digest and put it into
a pBlueskript SK II vector. This achieves two things, i you get your gene
with a Lamin sequence, a good kozak and an ATG in front of it, ii you get
a Sal I site in front of your gene. Now you simplt cut your gene out of the
blueskript vector using a SalI/NotI cut and ligate it into the pAdTRACK 1
shuttle vector. If this is done you are ready to procede to the next step.
2. Recombination
The recombination was a very tricky
part in the start, but do not worry now it's awalk in the part. All you have
to do is to go to the minus 80 freezer and get a tube of electrocompetent
BJ ADEASY bacteria, there is a paper box in the bottom trawer. Now transform
your shuttle construct into this cells using standard electroporation technique.
Thats it. You linearize here with PmeI,
use around 0.2m
g. You select with kanamycin and if you wish you can add additional
streptomycin to the plates, the bacteria carry a resistance against streptomycin.
By the way if you ever happen to have to make fresh competent cells grow
the BJ ADEASY you grow them in LB containing Streptomycin and Ampicillin.
Pick some colonies, I would recommend 5-10, isolate your DNA using alkaline
lysis miniprep, not Wizard or other kits using columns. Of the DNA
isolated take at least 5m
l (this is a low copy plasmid and the BJs don't contain much DNA either so
the yield is low) and digest with Pac I, BamH I and XhoI (seperatly of course)
and analyze the whole digests on a gel (0.7-1%). You should see the following:
Pac I 2 bands one around 40 kB and one around 3kB or 4.5 kB; Bam HI
21, 11 and 5 kB; XhoI 2x 20, 3, 1.5 and 0.7kB and you should see a band corresponding
to your insert. The confirmed recombinants you transform into DH1
a using the standard procedure
and isolate the DNA. If you have confirmed your construct you make a MIDI
prep using the Qiagen Midikit (take care low copy plasmid) and procede to
the next step.
3.Transfection
I use standard CaPO
4 here because in my hands it worked
the best with this DNA and cells. To do so, you first have to get 293
cells (take care we have also 293T cells and those do not work at all) into
culture. This cells are easy to handle and are happy in DMEM with 10% FBS.
To make the transfection you first seed about 1000000 cells into a 60mm petri
dish and let them sit over night. Parrallely you digest 5
mg of construct with PacI I usually
do this over night to ensure complete digestion. Next morning you precipate
the DNA with Ethanol and wash twice with 70% Ethanol, after this the tubes
should be handled in the hood because the DNA is considered sterile at this
point, dry the DNA briefly but do not overdry, you have a hard time to get
your DNA in solution. Take up your DNA in 50m
l 0.1x TE. Do not vortex!!!!
Now you label two sterile eppis 1 as #1,
the other as #2 or however you like it.
To tube #1 you add 169
ml of sterile water and 5
ml of 2.5M CaCl
2, mix gently.Now add, drop by drop, the 50
ml of the DNA and additional 26
ml CaCl2.
Mix gently.
Into tube #2 pipet 250
ml of 2X HEBS buffer solution.
(all m here mean of corse microliter)
Using a 1ml pipette connected to a pipet
aid start bubbling air into tube #2. While bubbling add the the content of
tube #1 dropwise. Continue to bubble for additional 5 seconds to ensure a
good mixing. Wait for one minute and than spread the solution over the cells.
Put your cells back into the incubator. You can follow the transfection efficiency
easily with the GFP marker. After 5-7 days you will observe little comet
like clusters of GFP expressing cells. Harvest the Virus after 7 days.
4.Harvest the Virus
This is easy. Scrabe your cells from the plate (do not use trypsin) and take them into a Falcon tube. Spin them down and take them up in 2 ml of cold PBS. Now you lyse the cells by performing four FrezzeThaw cycles. I freeze them in liquid nitrogen and thaw them in the 37 C waterbath, take care they should not warm up. After each cycle vortex vigourously. Spin down the cell debris and use the supernatant to infect two 60mm petri dishes containing 50-70% confluent 293 cells. Harvest the virus when appr. 50% of the cells have detached (after 3-5 days) using the same method as above.
5.Amplification of the virus (preperation of high titer viral
stocks)
1. Plate 293 cells in 150mm petri dishes so that they are at 90% confluency
at the time of infection. Usually I used twenty plates per virus to be amplified,
mark that you will have to use a lot of medium here, so better doublecheck
there is sufficient around.
2. Infect your cells at MOI around 5, if you have a titered stock If
you have to use a lysate prepared as outlined above I used half of
the lysate for all twenty plates, this turned out to be enough. You can follow
the infection under the fluorescence microscope, harvest the cells when all
of them have ronded up and appr. 50% have detached from the plate. I simply
washed the cells of the plates and span them down (5' at 500xg in a
benchtop centrifuge). Discard the supernatants (you can as well keep them,
lots of virus in there, but its not to stable. I tried once and the infectivity
declined pretty fast after storage at 4o
C, maybe one can freeze down aliquots.)
3. Resuspend the pellet in 8ml cold and sterile PBS. Perform four freeze/thaw
cycles as above and clear the lysate in Sorvall HS4 rotor at 7000xg for five
minutes.
4. Weigh 8,8 g of CsCl in a 50 ml tube, transfer 8.0ml cleared lysate to
the tube and vortex . transfer the the CsCl solution (~10ml, desnsity 1.3g/ml)
to a 12ml
Polyallomer tube for SW41 rotor, overlay with 2ml mineraloil.
Don't forget you need a balance tube. Spin the gradient at 32000 rpm, 10
oC, 18-24h.
5. Collect the virus fraction (~0.5-1ml) with 3ml syringe and an 18gg needle.
Mix with an equal volume of 2x storage buffer (10mM Tris pH8.0, 100mM
NaCl, 0.1% BSA, 50% Glycerol), filter sterilize and store at -20.
6. Check virus titer. Here you have different choices in the original protocoll
the recommended to make a dilution series and count the infected cells after
18 h under the fluoresence microsope.I found that rather tedious and propably
error prone. I switched to measure OD260.
To read OD, add 15 µl virus to 15 µl blank solution (Blank Solution
= 1.35g/ml CsCl mixed with equal vol 2X Storage Buffer) plus 100 µl
TE/0.1% SDS; vortex 30 seconds, centrifuge 5 min. measure A260. One
A260 unit contains ~1012 viral particles (particles:infectious particles =~20:1).