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The HSV Manual (v1.10) General notes about my HSV vectors: My HSV vectors are derived from herpes simplex virus 1 (HSV-1). Replicationdeficient virus is packaged via the amplicon system and purified on a sucrose gradient Inserts up to at least 12 kb can be packaged without significant loss of titer. “Short term vectors” vs. retrograde “long term” vectors: Significant expression from my “short term” vectors is seen in vivo at the site of injection within 2-3 hours, with maximal expression from 3-5 days post-injection. In addition, expression only lasts about 8 days in vivo, making the short-term vectors ideal for A-B-A experimental designs. The retrograde “long term” HSV is expressed in retrograde neurons beginning 13 weeks post-injection, and expression in these neurons persists for months. Retrograde HSVs with the hEF1a promoter are expressed only sparsely or not at all at the site of injection. 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 our 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. Versatility of HSV vectors: Over 100 different HSV backbones are available for the myriad uses of these vectors in vivo and in vitro. They fall into the following general categories. 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. Co-expression 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 HSVs (long term only): This is a completely new plasmid set with optimized transcription elements that enable long term expression in retrograde neurons. The expression profile and spatial distribution are quite different from those 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). Expression in these neurons is persistent, lasting at least 3 months. My most effective retrograde vectors are those driven by the hEF1α promoter. Using HSV vectors in vitro: Viruses typically express detectable amounts of protein in as little as 2-3 hours in vitro in cell culture. The hSyn promoter is recommended for in vitro studies in primary neuronal cultures. 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 for the life of the culture. 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. 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 use of these viruses in animals, because HSV can be transmitted only by contact of one wet surface with another, and cannot be transmitted via aerosols. There is no need for “special” housing for animals that have been injected with HSV, because the virus is replication-incompetent, so that the animals do not “shed” virus post-injection. HSV can be inactivated (“killed”) with 70% ethanol or bleach. Dispose of unused virus in “Biohazard” waste bins. Preparation of HSV plasmid DNAs for packaging: I have very specific criteria for submitting HSV plasmids for packaging into virus. Plasmids that do not exactly conform to these criteria will not be packaged. I use 2ug per virus prep, but please send at least 20ug of plasmid so that I can make additional batches of virus if needed. 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 the exact DNA concentration. A very general 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. Quantities and Titers HSV “FULL PREPS”: -ST HSV: min volume and titer 400 ul @ 1 x 109 infectious units/ml -HT viruses (retrograde viruses only): minimum volume and titer 200 ul @ 2 x 109 infectious units/ml -5X viruses (retrograde viruses only): minimum volume and titer 80 ul @ 5 x 109 infectious units/ml HSV ALIQUOTS: -ST HSV: min volume and titer 30 ul @ 1 x 109 infectious units/ml -HT viruses: minimum volume and titer 30 ul @ 3.5 x 109 infectious units/ml -5X viruses (rabies and TVA only): minimum volume and titer 20 ul @ 5 x 109 infectious units/ml Source for EnvA pseudotyped rabies virus to use in concert with the HSV vectors expressing rabiesG and TVA: Ed Callaway says: "There are 2 options for obtaining EnvA pseudotyped rabies virus for your experiments. You can either purchase purified virus ready for brain injections from the Salk Vector Core (http://vectorcore.salk.edu) or my lab can provide cell lines and starter stock for you to grow the virus yourself. If you would like to grow your own then please see Osakada and Callaway Nature Protocols 2013 for detailed instructions and a list of the reagents you would need. Once you know what cell lines and starter virus stocks you need please send an email to me and to Dhru Chatterjee and we will prepare an MTA and arrange for shipping of reagents. In order to recover some of the costs involved in generating the reagents we charge $200 for each cell line and for each starter stock that is provided" Source for cre-and flpo-dependent AAV viruses that can be used in combination with the retrograde HSV viruses in the INTRSECT protocol: Cre-and flpo-dependent AAV viruses that can be used in combination with my retrograde HSV viruses may be requested directly from Dr. Karl Deisseroth and Charu Ramakrishnan The PI of your lab should make that request, and an MTA will be necessary. Information is available at this site: http://web.stanford.edu/group/dlab/optogenetics/request_dna.html An important note about potential XFP toxicity: Over the years, I have continually optimized my HSV packaging protocols to produce the highest titers possible. This gradual increase in titer has benefited 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 immediately around 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 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. There are no toxicity problems with hEF1a retrograde viruses, no matter how high their titers, because they are expressed only sparsely or not at all at the site of injection.