The HSV Manual (v1.6)
General notes about my HSV vectors:
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 only lasts about 8 days in vivo, making it ideal for A-B-A experimental
designs. Inserts up to at least 12 kb can be packaged without significant loss of titer.
Different HSVs:
We currently have several options for increased flexibility in making and using
HSVs:
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HSV PrPUC (short term): This is my vanilla HSV plasmid, in which
expression is driven by the HSV IE 4/5 promoter.
HSV p1005 (short term): 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): 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): 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
CaMKII or hEF1α promoters.
Taking care of your new HSVs:
The viruses are in PBS+10% sucrose+25 mM HEPES, 7.3. HSVs are highly
perishable and do not do well at room temperature or at 4. Keep your HSVs at
80 or on dry ice until you use them. 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 37 o 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
Individuals who aliquot very low volumes (anything under 20 ul) 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 lowbinding 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:
Viruses typically express detectable amounts of protein in as little as 2-3 hours in
vivo and in cell culture. Maximal expression 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 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 48- and
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.
 Although fume hoods are not required for these viruses (HSV can be
transmitted only by contact of one wet surface with another, and cannot be
transmitted via aerosols), individual users may feel more comfortable
using BLII hoods for surgery as well as cell culture.
 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 100ng/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 2.0 ul of virus into rat (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. 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 in PBS
immediately prior to injecting it. Try dilutions ranging from 60% (6 µl of virus, 4 µl PBS)
to 75%. It will work. Diluting the virus is not recommended if the virus will be used for
retrograde transport, since you probably will want to express the transgene in as many
retrograde neurons as possible.