Resources

Optimising Protein Expression

Once you have finished making your recombinant baculoviruses expression vector you naturally want to maximize the amount of protein made in insect cells.  A point we have emphasized in a previous blog is the importance of determining the infectious titre of your virus stock accurately.  It is also prudent that you produce a stock of virus large enough that you can conduct a series of experiments to optimise protein production

Longevity of Infectious Budded Baculovirus Stocks

A question that comes up repeatedly is the long term stability of recombinant budded baculovirus stocks.  This is a particular issue now that most of them are generated using insect cells grown in serum-free medium.  It has long been a mantra in virology that you should always have a little bit of protein in your virus suspension to help stabilise the virion structure or prevent non-specific binding to glass or

Rapid Virus Titration by QPCR

Knowing the infectious titre of a recombinant virus stock is really important prior to testing for protein production in insect cells.  It enables you to add the optimal quantity of virus to initiate virus infection.  Too much virus and you are wasting your stock.  Too little virus and you won’t establish a synchronous infection.  For some proteins this may not matter, as the virus produced by the initial round of

Virus Like Particles VLPs for Therapeutics and Research

Oxford Expression Technologies (OET) is now able to apply its flashBAC™ baculovirus expression system to vaccine production, particularly in the exciting new field of virus like particle (VLP) vaccines. A virus like particle is an assembly of virus structural proteins that mimics the configuration of a real virus, except that it contains no genetic material. If a person is vaccinated with VLPs then an immune response is generated as if

Titrating Stocks of Recombinant Baculoviruses using the Plaque Assay Technique

We were going to devote this blog to a comprehensive account of how you might determine the infectious titre of your virus stock.  However, when our CEO got wind of this he started ruminating about how things were “back in the day” and how he had to collect seaweed from the seashore to extract agar for his overlays…!  No, we didn’t believe him either, but he is approaching his 7th

Production of High Titre Virus Stocks

In our last blog post we talked about the process of transfecting insect cells with virus DNA and how you could recognize success or otherwise.  Transfections are normally done on a fairly modest scale (2-5ml) in culture dishes.  Therefore your P0 virus stock is of low volume and probably modest infectious titre (107 plaque forming units [pfu] per ml or lower; we will discuss how to measure this value in

Transfection of Insect Cells with Baculovirus DNA

Independent of the method you use to produce recombinant viruses, at some point you have to transfect insect cells with virus DNA to reconstitute infectious virus particles.  When using the flashBAC™ system, you simply add it to your transfer vector containing the foreign gene and use a suitable transfection agent (e.g. baculoFECTIN II )to achieve entry of DNA into the cells.  The transfected cells are then incubated at 28°C for

How Healthy are your Cells?

Hopefully after the seasonal break your insect cells are still in good health.  Recognizing healthy cells seems a no brainer, but if you haven’t got much experience with insect cells it can be more of a problem.  Experts often talk about “shiny appearances” and “well defined membranes”, which is fine if you know what they mean but puzzling if you are in your first foray into cell culture.  So to

To Heat Treat or Not to Heat Treat Calf Serum, that is the Question?

Ok, so the Bard is probably spinning in his grave at our blatant plagiarism of the script from the “Scottish Play” (it is bad luck to use its real name, apparently).  However, it seems an apt way to introduce the question of whether or not you should heat treat foetal bovine serum (FBS) before using it to propagate insect cells.  Of course, Spodoptera or Trichoplusia spp. cells are mostly grown

Cells at Christmas

So, you have had the Christmas Party and hopefully are looking forward to the holiday season.  Many labs shut down between Christmas and New Year and you may not return until January 4th.  In all the build up to the festive season have you remembered everything?  Aunt Hilda’s bottle of lavender water and Uncle Horace’s box of cigars may be already wrapped and under the tree but aren’t you forgetting

Cell Culture Passage History and Stress

No, not how you feel when the cells don’t behave! This relates to keeping your cell cultures in a happy state. Cell passage history Each time you sub culture your cells you probably faithfully record on the new flask various bits of information such as the name of the cell line, date, the medium used and passage number.  This last item ideally relates to the number of times the cells

Are they Growing? – Beginner’s Guide to Cell Culture

Cell culture may be one of the most routine laboratory tasks, but many times it also proves to be extremely frustrating. Cells grown in the labs are living organisms, and they seem to have the mindset of a moody teenager – happy one moment and extremely upset the very next one. Unfortunately (or fortunately) unlike teenagers they are not able to throw a tantrum and scream out what bothers them.

Relevant Papers from 2000-2009

2000 King, LA and Possee, RD. (2000). Insect cell culture for baculovirus replication. Encyclopaedia of Life Sciences, Macmillan Press London. In press. Possee,RD and King, LA. (2000). Baculoviruses Encyclopaedia of Life Sciences. Macmillan Press, London. In press. Thomas, CA, Gooday, GW, King, LA and Possee, RD. (2000). Mutagenesis of the active site coding region of the Autographa californica nucleopolyhedrovirus chitinase gene. J. Gen. Virol. 81, 1403-1411. Grasela, JJ., Mcintosh, AH.,

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