baculovirus / insect cell / strange "crystals"?

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    • #8614
      nexus2006
      Participant

      Dear All,

      I have a question concerning the baculovirus expression system. I attached a picture of
      SF9-cell infected with my virus, it shows strange "needle"-like
      structure, they only appear in infected cells, i do controls with just
      the lipofection agent and the bacmid DNA alone. It looks like
      contamination to me, but it just appears roughly 2 days postinfection
      and does not "overgrow" the culture.
      I don’t know if you maybe have an idea or know somebody who has, i
      will be thankful for any answer.
      Regards

      Rudi K.


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    • #78124
      h2so4hurts
      Participant

      What are they infected with? I’ve never seen structures like that appear in my infections.

    • #78130
      biohazard
      Participant

      It’s been a while since I worked with baculoviruses, so unfortunately this isn’t too fresh in my memory. However, the first thing that comes to mind is that it looks like a crystal, and if so, then it’s likely to be either the virus or the protein you’re expressing (are you infecting with AcMNPV by any chance?) – both have tendency to form crystals or inclusion bodies under certain conditions, in certain expression systems… although I have to admit their morphology couldn’t be further from the polyhedron you expect to see, so that obviously isn’t what we see here 🙂

      It could also be something that crystallises in the medium, but that sounds quite far fetched, especially if it only takes place in the infected cells. I’ll think this little further, but at least currently I’m guessing they’re crystal spikes of some sort.

      If this really disturbs you, I might be able to show the pic (it’s quite nice addition to your post!) and the question itself to few people that have worked with baculoviruses for a long time. They may or may not be able to help 😉

      This is actually quite interesting…!

      EDIT: I took the liberty to forward your question and the pic to some baculo experts, let’s see if they know what this is all about.

    • #78177
      nexus2006
      Participant

      thank you very much for the inspiring hints!
      It is a AcMNPV encoding a protease which has a secretion signal. I also thought of crystals, protein or salt. we will do some TEM just for fun, to see if they are microorganisms or not.

      @biohazard
      : maybe one of your guys have seen those structures, i really want to know what it is 😉

    • #78375
      biohazard
      Participant

      Well, didn’t hear anything from our baculo guys so far, let’s see if we have better luck next week. Let me know if you manage to solve this meanwhile 🙂

    • #78912
      biohazard
      Participant

      Apparently the persons I asked are either too busy to answer, or don’t have any clue either… shame, it would’ve interesting to know what those structures are.

      Anyway, if I hear something, I’ll post it here.

    • #79143
      nexus2006
      Participant

      Thanks anyway for your effort, i made now some TEM pictures and also from SDS-Page the structures appear to be protein crystals… now i am looking for a protocol to separate them from the cells 🙄

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