On another note, we have moved from baculovirus to stable poly or
monoclonal Tn5 cell lines using the pIB/V5-HIS vector. It removes all
concerns about titering and viral propagation, and for most of our
proteins, the expression level is the same or better. We secrete all
our proteins, so the stable cell line is great, you just grow several
liters of the cells to a high density, harvest the supernatant, and
purify. It's much simpler.
Nat
On Thu, Mar 31, 2011 at 3:52 PM, Chun Luo <[log in to unmask]> wrote:
> As many have mentioned, there is no need to titer baculoviruses for protein expression purpose. Plaque assay or other titering methods can give >10-fold variation between two operators. Cells can be different from time to time as well. Sf9 cells in different labs are quite different. MOI reported in literature is pretty much meaningless. I think most authors and especially their labmates agree on this point. The important thing is how insect cells are infected. In this aspect, industry is at least 10 year in advance than academia. We have developed methods to quantify the infection and ensure high quality virus production and protein expression. Nat has described some steps how to do it.
> When properly made and stored, recombinant baculoviruses are stable for at least a year without the need of adding FBS or other stabilizer. We have used several 2 year old viruses and got the exact same results as when they were freshly made. TIPS is one great way for long term virus storage, but reqires expensive equipment and know-how. Bacmids can be stored cheaply (or plasmids if not using Bac-to-Bac system). It takes only a week to generate enough "high tiger" viruses sufficient for 5-10L expression. For most academic research, there may be no need to do any viral amplification.
> Cheers,
> Chun
> Accelagen
>
> -----Original Message-----
> From: Nathaniel Clark <[log in to unmask]>
> Sender: CCP4 bulletin board <[log in to unmask]>
> Date: Thu, 31 Mar 2011 13:20:14
> To: <[log in to unmask]>
> Reply-To: Nathaniel Clark <[log in to unmask]>
> Subject: Re: [ccp4bb] titering baculovirus ?
>
> We don't do anything special to store the virus, just a dark fridge.
> You do need to mix well as the virus can settle.
>
> You might want to try this 'TIPS' approach. Basically you freeze
> baculovirus infected cells, and use them as your virus innoculum. I
> think I tried it once and it didn't really work, but I didn't really
> try too hard...
> There is a research paper on the subject that I can't find, but here
> is the first thing I found on google:
> http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Wasilko-PERG2007.pdf
>
> Nat
>
> On Thu, Mar 31, 2011 at 1:12 PM, chitra shintre
> <[log in to unmask]> wrote:
>> Hi,
>> We have observed this virus "going off" within 2 months as well. ...any idea
>> why this happens?
>> Chitra
>>
>> On 31 March 2011 18:07, Nathaniel Clark <[log in to unmask]> wrote:
>>>
>>> We do adherent if we have a small volume of low titer virus, but as
>>> soon as we have a decent titer we will start with a 30 ml suspension
>>> flask. Normally I add something like 0.5 ml, which is probably more
>>> then I need. We only use serum free media right now. As I mentioned,
>>> if you need to boost the titer of a low titer stock, we plate a t25
>>> flask with Sf9s, take off the media, and incubate it in 1 ml of pure
>>> virus (or two-fold diluted) for an hour or so, then remove (and save)
>>> that virus stock and give the cells fresh media.
>>>
>>> For fold dilution, just do a rough calculation of titer. Once you are
>>> at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate
>>> the ballpark volume to infect a given number of cells at an MOI of 1.
>>> From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you
>>> do an amplification that doesn't work, decide if you were to high
>>> (MOI=>10 not great for amplifying) or too low (MOI=<0.1) and try
>>> again. Better yet, just set up 3 amplifications at the same time,
>>> whichever worked the best, use for another round of amplification, or
>>> for infections.
>>>
>>> For duration, we do 3 days amplification (post-infection), but
>>> sometimes we let them go a few days longer, which allows a secondary
>>> infection of the cells that weren't infected by the viral innoculum.
>>> There doesn't seem to be any harm to letting infections go longer; at
>>> some point all the cells die, but since the virus is pretty stable,
>>> it's no problem. We have a CEDEX cell counter which tells you average
>>> cell diameter, which is a great read out for infection. Also looking
>>> and density and viability versus an uninfected control will tell you
>>> if the infection worked.
>>> Nat
>>>
>>> Nat Clark
>>> Graduate Student
>>> Garman Lab
>>> Biochemistry and Molecular Biology Dept.
>>> UMass Amherst
>>>
>>> On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein
>>> <[log in to unmask]> wrote:
>>> > A related question: how do most people amplify their baculovirus
>>> > stocks? Adherent cultures vs suspension? Fold dilution at each stage
>>> > (P1 to P2, P2 to P3)? Duration of each amplification stage?
>>> >
>>> > We have some viral stocks that "go off" rather quickly (1-2 months)
>>> > despite being stored with FBS in a cool, dark place.
>>> >
>>> >
>>> > Katya
>>> >
>>> >
>>> > On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett <[log in to unmask]>
>>> > wrote:
>>> >> Though I'm not meaning to turn this into a plaque assay burn session,
>>> >> for
>>> >> many reasons we have also abandoned it and instead titer our
>>> >> baculovirus
>>> >> stocks (P3 and P4) using flow cytometry. We use an antibody against
>>> >> viral
>>> >> gp64 that has been labeled with PE and stain infected cells in 96 well
>>> >> plates with the PE-antibody. We then measure PE fluorescence on a flow
>>> >> cytometer (you can also do cell counts, viability determinations,
>>> >> etc.). We
>>> >> equate 1 fluorescent event as 1 infected cell. Since we know how many
>>> >> cells
>>> >> have been plated in each well, we can determine the percentage infected
>>> >> in
>>> >> each well. We calculate a "non-normalized" titer from this data alone
>>> >> or we
>>> >> compare this data to a standardized virus and determine a normalized
>>> >> titer
>>> >> using a standard curve. From infection to having a titer in hand takes
>>> >> about
>>> >> 24 hours. Of course, the potential bottleneck is access to a flow
>>> >> cytometer!
>>> >> I can give more experimental details off-board.
>>> >> I should say that for getting an idea of relative titers and to test
>>> >> protein
>>> >> expression on a small scale, we also do the "effective titer" tests as
>>> >> suggested by Nat, with cell morphology and immunoblots as our read-out
>>> >> of
>>> >> virus potency and recombinant protein expression, respectively. No
>>> >> doubt,
>>> >> this will get you a long way but at some point, I argue, you need to
>>> >> determine an actual, absolute titer for duplication of results,
>>> >> troubleshooting, monitoring virus health over time, publications, etc.
>>> >>
>>> >> HTH-
>>> >> Brad
>>> >>
>>> >> On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms]
>>> >> <[log in to unmask]> wrote:
>>> >>>
>>> >>> I checked with someone in our protein production group and got the
>>> >>> following response:
>>> >>>
>>> >>> We also stopped doing the virus titration with the plaque assay and
>>> >>> instead are performing expression test with different concentration of
>>> >>> virus
>>> >>> from the 3rd amplification. But for some viruses we still have doubts
>>> >>> concerning the amplification success, so we are now evaluating a new
>>> >>> technology using qPCR with the following kit
>>> >>> (http://oetltd.com/products/category/baculoquant/). So you might have
>>> >>> a look
>>> >>> and see if it could be useful for your group. We would also be curious
>>> >>> to
>>> >>> hear if anyone else has experience with this approach.
>>> >>>
>>> >>> I hope this helps.
>>> >>> Jay
>>> >>>
>>> >>> -----Original Message-----
>>> >>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
>>> >>> Nathaniel Clark
>>> >>> Sent: Wednesday, March 30, 2011 11:38 PM
>>> >>> To: [log in to unmask]
>>> >>> Subject: Re: [ccp4bb] titering baculovirus ?
>>> >>>
>>> >>> We don't have a problem getting them to stick to the plates in
>>> >>> serum-free
>>> >>> media, or in 5% FBS media. The more challenging part is getting the
>>> >>> plating
>>> >>> density just right, too low and the plaques are too big, to high and
>>> >>> they
>>> >>> are too small. Or if the cells dry out, or if your agarose overlay is
>>> >>> too
>>> >>> hot, etc...
>>> >>>
>>> >>> However, we have actually stopped titering all together. We find
>>> >>> early
>>> >>> stocks (from co-transfection, or plaque purification) are 'low', but
>>> >>> after
>>> >>> ~2 rounds of amplification in adherent culture of the 'low'
>>> >>> titer stock(using a large volume of low-titer virus in a t25 flask),
>>> >>> we
>>> >>> can add ~0.5 ml to a 30-100 ml shake flask of Sf9's (serum free
>>> >>> media) and get a high titer stock( ie. >10^8 pfu/ml). From there we
>>> >>> amplify with ~ 10 mls of virus in a 1 L shaker culture, and we have
>>> >>> our
>>> >>> large volume high-titer stock. Sometimes we will incubate the cells
>>> >>> in pure
>>> >>> virus stock in a t25 flask for 1 hour, take the virus off, and add
>>> >>> fresh
>>> >>> media, as a way to rescue low-titer stocks.
>>> >>>
>>> >>> If you are just trying to titer (not plaque-purify), you can just take
>>> >>> 10
>>> >>> fold dilutions of your virus, and do several small scale infections
>>> >>> in 6 well plates, 10 ml shaker cultures, whatever you prefer. At the
>>> >>> lowest virus concentration where you see a synchronous infection
>>> >>> (judged
>>> >>> by protein expression levels, or cell-diameter if you have a cell
>>> >>> counter,
>>> >>> or by viewing with a trained eye), you call that an MOI=1. From there
>>> >>> you
>>> >>> know the number of cells in the plate, and the volume of virus you
>>> >>> added, so
>>> >>> you can calculate an effective titer.
>>> >>> Plaque assays are really difficult and slow, and if you are just
>>> >>> trying to
>>> >>> make protein, an effective titer is fine, the absolute number isn't
>>> >>> that
>>> >>> helpful, Nat
>>> >>>
>>> >>> Nat Clark
>>> >>> Graduate Student
>>> >>> Garman Lab
>>> >>> Biochemistry and Molecular Biology Dept.
>>> >>> UMass Amherst
>>> >>>
>>> >>>
>>> >>> On Wed, Mar 30, 2011 at 5:14 PM, Gloria Borgstahl
>>> >>> <[log in to unmask]>
>>> >>> wrote:
>>> >>> > Hi Guys,
>>> >>> > we are learning to work with Sf9 cells and Carol in my lab wanted me
>>> >>> > to ask you the following question. Many thanks for any help, G
>>> >>> >
>>> >>> > I need to titer a baculovirus stock in my suspension-adapted Sf9
>>> >>> > cells. I know that these can be encouraged to attach better to
>>> >>> > tissue culture plastic if they have added FBS (about 10%), but am
>>> >>> > not
>>> >>> > sure that they will not be migrating and hiding plaques. Does
>>> >>> > anyone
>>> >>> > have suggestions about how to keep them more firmly anchored during
>>> >>> > the baculovirus titration, or about another cell line that we could
>>> >>> > use
>>> >>> > instead?
>>> >>>
>>> >>>
>>> >>> This message has been scanned for malware by Websense.
>>> >>> www.websense.com
>>> >>
>>> >>
>>> >
>>
>>
>
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