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The HSV Manual (v1.9)
General notes about my HSV vectors:
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My HSV vectors are derived from herpes simplex virus 1 (HSV-1).
Replication-deficient virus is packaged via the amplicon system and purified
on a sucrose gradient. HSV has advantages over many other viral vectors
(see, e.g., Neve et al., BioTechniques 2005; 39:381-391).
Significant expression from my “short term” vectors is seen in vivo within 2-3
hours, with maximal expression from 3-5 days post-injection. In addition,
expression at the site of injection only lasts about 8 days in vivo, making it
ideal for A-B-A experimental designs.
In the case of my retrograde vectors (hEF1α promoter), expression at the site
of injection is transient or occurs not at all. 1-3 weeks later, expression
appears in neurons that project to the site of injection (retrograde neurons)
and persists for at least several months.
Inserts up to at least 12 kb can be packaged without significant loss of titer.
Different HSVs:
I currently have several options for increased flexibility in making and using
HSVs:
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HSV PrPUC (short term only): This is my vanilla HSV plasmid, in which
expression is driven by the HSV IE 4/5 promoter.
HSV p1005 (short term only): This is a modified HSV amplicon plasmid with
an added transcription cassette expressing GFP; i.e., it produces a separate
transcript (separate promoter and poly(A)) for GFP. The target gene is still
driven by the IE 4/5 promoter, while the GFP is driven by a CMV promoter.
The two transcription cassettes are in a nose-to-tail orientation. Coexpression is generally >90%. I have available p1005 vectors that co-express
GFP, DsRed2, EYFP, mCherry, tdTomato, synaptophysin-EYFP or
synaptophysin-mCherry.
HSV p1006 (short term only): This is a modified version of p1005 in which the
GFP cDNA is replaced with a MCS, so that two transcriptional cassettes are
available for the expression of more than one transgene. It is also useful for
cloning of transgenes that may be toxic during the packaging process when
their expression is driven by the IE 4/5 promoter. This is especially true for
XFPs or XFP fusions, which are not toxic during the packaging process when
they’re cloned downstream of the CMV promoter.
Retrograde HSV (long term only): This is a completely new plasmid set with
optimized transcription elements so that in vivo expression is long term ( > 3
months, with no diminution of expression). The expression profile and spatial
distribution are quite different from that of the short term vectors. Expression
at the site of injection is transient or occurs not at all. 1-3 weeks later,
expression appears in neurons that project to the site of injection (retrograde
neurons). My most effective retrograde vectors are those driven by the
hEF1α promoter.
Taking care of your new HSVs:
The viruses are in PBS+10% sucrose+25 mM HEPES, 7.3. This is a new buffer
that I recently made, that confers stability to the viruses. While I recommend the
usual aliquotting and -80 C storage, it’s not the end of the world if your freezer
fails. If you get the virus back into -80 C within a day, it probably will not have
lost any titer. Avoid repetitive freeze-thaws, as this reduces titer slightly. The
thing to remember with HSV, every time you do a freeze-thaw cycle, is: thaw fast
(37o C water bath) and freeze fast. The very first time that you thaw your new
HSV prep (rapidly, in a 37o C bath), you must aliquot it into useable volumes
(thereby avoiding extra freeze-thaw cycles). I now use Corning low binding
microcentrifuge tubes to aliquot my viruses:
Costar Cat. No. 3206 for 0.65 ml tubes
Costar Cat. No. 3207 for 1.7 ml tubes
Use of these tubes is particularly recommended for Individuals who aliquot very
low volumes (anything under 20 ul), who lose virus to the walls of the tube during
a thaw due to the large surface area:volume ratio. I made two improvements to
my HSV protocol, to solve this problem. First, I am now including 25 mm
HEPES, 7.3 in the virus preps because it turns out, unexpectedly, that the drop in
pH of the PBS vehicle when it is frozen causes increased binding of the virus to
the walls of the tube. Secondly, even with the altered pH, there was still room for
improvement, so I tracked down some low-binding microcentrifuge tubes (link
above). Voila! I can now do a freeze-thaw on a 5 ul aliquot with no detectable
loss of virus. These improvements are especially useful for those doing
retrograde tracing experiments with my HSVs, because they need to inject as
much virus as possible in order to see robust retrograde transport.
So please, when you aliquot your viruses, use the low binding tubes. I am using
them as well.
Using your new HSVs:
The short term viruses typically express detectable amounts of protein in as little
as 2-3 hours in vivo and in cell culture. Maximal expression at the site of
injection in vivo is achieved at 24hrs post injection and stays high during days 2-5
post-injection. Expression drops about 50% on day 6 and is gone by 8-10 days
post-injection.
For in vitro work, you can get around the occasional toxicity problem that is
caused by high expression of fluorescent proteins, by infecting for a relatively
short period of time or infecting with fewer viral particles. We know by
immunoblots that in cultured neurons we can detect expression from the virus
transgene within 2 hours of infection. Expression peaks between 6 and 9 hours
after infection, and stays at that level indefinitely. We frequently will harvest or fix
cells for assays at 5-6 hours post-infection. On the other hand, we also do 48and 72-hour experiments. If the cells infected with the virus are mitotic, it is best
to do the experiment within 24 hours, since the virus will be diluted in the
population as the cells divide (it is episomal and does not replicate along with the
cellular DNA). For primary neuronal cultures, infect the cultures at an moi of 2 for
>95% infection, or an moi of 1 for 70-80% infection.
To infect neuronal or conventional dividing cells in vitro, simply add the virus to
whatever medium the cells are in. You do not need to remove the viruscontaining medium and replace it with fresh medium at any point.
The GFP in the p1005 viruses is easily denatured and stops fluorescing well in a
matter of days or weeks in fixed tissue (depending on conditions). For dead
tissue, it is best to run IHC for the GFP protein (on the FITC/CY2 channel).
Safety Issues:
Our modified HSV viruses are replication-incompetent. As a result, the viruses
are very safe. However, as a precaution, all HSVs are to be treated as
hazardous. Here are some guidelines for use:
 Always wear gloves and a lab coat.
 Biosafety cabinets are not required for animal work with these viruses,
because HSV can be transmitted only by contact of one wet surface with
another, and cannot be transmitted via aerosols). If the virus is
accidentally spilled on a dry surface, it dies within seconds.
 Virus can be inactivated (“killed”) with 70% ethanol or bleach.
 Dispose of unused virus in “Biohazard” waste bins.
Making new HSV vectors:
I have very specific criteria for submitting HSV plasmids for packaging.
Plasmids that do not exactly conform to these criteria will not be packaged:
I use 2ug per prep, but please send at least 20ug of plasmid so that I can make
additional batches of virus if needed.
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DNA should be midi- or maxi-prepped using Qiagen kits. Miniprep DNA does not
transfect well.
Minimum concentration should be 200ng/ul.
Plasmids MUST be in TE (10 mM Tris, pH 7.5, 1 mM EDTA).
I need an EXACT DNA concentration.
Injection procedure for in vivo studies
The general procedure is to microinfuse up to 2.0 ul of virus into rat (up to 1 ul for
mouse) over a 10 min period using a syringe pump. The injector is left in place
for another 10 min to minimize the vacuum effect when it is removed, which will
draw the vector up the track made by the injector.
Lesions at the injection site can occur when bone debris generated while drilling
the skull is forced into the brain when lowering the injector. The hole in the skull
should be made carefully and cleaned thoroughly before putting the injector into
the brain. Everything needs to be done SLOWLY, and it can be difficult to resist
the urge to rush it.
An important note:
About four years ago, I made a major breakthrough in my HSV packaging technology
that increased the titers of my HSV constructs 3-5-fold. My titers had been in the range
of 6 x 10^7 to 1 x 10^8 transducing units/ml; they now range from 3 to 5 x 10^8
transducing units/ml. This huge increase in titer was great for most of those using my
vectors, who wanted greater spread and more robust expression in vivo. However, it
had the unintended consequence, in a very few regions of the brain, of causing XFP
toxicity at the site of injection. Note that the virus itself is not toxic. Try the virus at full
strength to start with. (I’m getting too many emails from people who start by diluting
the virus and then tell me that they want to see more cells infected.) If you see toxicity,
there is a very, very simple fix: dilute the virus with Dulbecco’s PBS with calcium and
magnesium (Mediatech number 21-030-CM) immediately prior to injecting it. Try
dilutions ranging from 60% (6 µl of virus, 4 µl PBS) to 75%. It will work. If your ST HSV
does not express an XFP or XFP fusion, you will not see toxicity, since the toxicity
arises from the XFP expression, not from the virus itself.
Diluting the virus is not recommended if the virus will be used for retrograde transport,
since you want to express the transgene in as many retrograde neurons as possible.
There are no toxicity problems with retrograde viruses, no matter how high their titers.