We’ve said it enough times; the plaque-assay is the ‘gold standard’ for determining virus infectivity. But this doesn’t make the procedure any easier! Whether it’s the agonising 5 day waiting period or the disorientating number of steps, plaque-assays can seem like a chore and we’ve heard of more than enough non-virologists admit to skipping this laborious technique in favour of a simpler solution. (more…)
In our previous blog we talked about the early stages of growing insect cells when you might just have revived a frozen stock and set up either monolayer or suspension cultures (or both). If you have established a viable suspension culture – and here we are talking primarily about insect cells such as Sf21 or Sf9 – you will probably monitor them fairly closely for their rate of growth. The ideal scenario is that your cells will double in number every 24 hours. However, frequently this isn’t the case. Why not?
A lot can depend on the seeding density of your cells after dilution from a previous culture. Another factor is the cell count attained by a culture before you sub culture or passage them to a fresh flask.
Let’s consider the first factor, seeding density. Most insect cells are grown in serum-free medium. This has permitted greatly increased cell culture densities to be attained compared to the days of using medium that contained serum. Anti-foam additives have also reduced shear forces, which develop from frothing and bubbles, so vigorous shaking can be used to increase the aeration of cultures. A slight disadvantage to this is that some cells, particularly Sf9s, are not happy if they are diluted too much on sub culture. We recommend a seeding density of no lower than 4-5 x 105 cells/ml. This can be particularly important in the first few passages of cells after they have been established from a frozen stock. It is very hard to define, but we have noted that cells freshly thawed can take a few sub cultures before they attain their maximum growth rate.
The second factor to consider is the density of your cells at the point of sub culture. Manufacturers of insect cell culture medium proudly claim that your cells can reach 107 or greater cells/ml in their product. This is great marketing material, but in day to day use you probably don’t want to force your cells to these densities on a routine basis. At the higher cell densities your culture will be approaching nutrient exhaustion and accumulation of waste by products. These can cause stress to your cells and result in the development of vacuoles and a grainy appearance. Your cells may still be close to 100% viable, but their growth rate will slow and the stationary phase of a culture is reached. This has the consequence that when you eventually sub culture your cells they may not start dividing immediately they are placed into fresh medium. If you monitor their growth you may see a lag before they begin to increase in number.
A further consequence of setting up a new culture of cells from one that had attained a very high density is that your dilution factor will be quite high. We have noted that dilution factors greater than 10 tend to result in slower initial growth of cells. The reasons for this are unclear but are probably a combination of the cells being in the stationary phase at passage and the low concentration of “conditioned” medium in the new culture. Conditioned or used medium is thought to contain factors generated by cells grown in the absence of serum that help other cells divide efficiently.
So ideally you should never grow insect cells in suspension culture to very high densities. We think it is best to let them reach about 3-6 x 106 cells/ml and then passage them to a new seeding density of 4-5 x 105 cells/ml. However, if your cells are only recently established from a frozen stock it may be wise to increase this seeding density slightly to 106 cells/ml to ensure they start dividing quickly.
More information of growing insect cells may be found in our baculoCOMPLETE manual: A Complete Laboratory Guide to the Baculovirus Expression System and Insect Cell Culture. If you do experience difficulties growing insect cells please contact us via firstname.lastname@example.org and we will try to help you.
A frequent customer query concerns baculovirus transfer vector plasmid compatibility with either flashBACTM or other systems for making recombinant viruses. Obviously, if you are buying flashBACTM and our pOET range of transfer vectors you won’t have a problem. However, given that baculovirus expression vectors have been around since 1983 and many labs have produced variations around a theme, it is not surprising that confusion can arise. (more…)
While OET is currently unable to offer GMP manufacture of recombinant proteins or viruses, we can offer a premium service for pre-GMP manufacture. This can serve as a very useful bridge to full GMP projects conducted by other service providers. So what is the advantage of a pre-GMP step? (more…)
A question that comes up from time to time is the use of co-infection with baculovirus vectors for multiple protein expression in insect cells. The need arises if you have produced a number of baculovirus vectors that each synthesise a component of a multi-subunit complex, such as an enzyme or a virus like particle. Intuitively, you know that it is probably necessary to add many virus particles per cell to achieve efficient infection with each recombinant. But how many viruses can you add to each cell and still get each one into every cell in equal numbers? (more…)
A question we are asked from time to time is to identify baculovirus transfer vectors
compatible with our flashBAC™ system. We obviously nominate our own extensive range that is available but frequently someone has an old plasmid from years ago that they want to reuse. (more…)
Variable expression of multi-component protein complexes is increasingly necessary as more sophisticated projects are attempted. But how can this be achieved? Most of our expression work is based on the baculovirus system, in particular our flashBAC™ vectors that permit easy recombinant virus generation in a one-step process. These insect-specific viruses offer an ideal platform for producing a wide range of recombinant proteins including nuclear, cytoplasmic, membrane-bound and secreted examples. (more…)
Between initial tests and larger scale up you may want to conduct intermediate scale recombinant protein production using baculovirus vectors. If you are working with several constructs then this can begin to get expensive of cell culture vessels such as the ubiquitous 125ml conical flasks, in which you can amplify 25-30mls of virus-infected cells. Space on shakers can also be at a premium in some labs. (more…)
Oxford Expression Technologies are the experts when it comes to the science of baculovirus. For almost 10 years now we have specialised in recombinant protein production in insect cells and the ‘Bac to the Future’ blog is the culmination of this extensive knowledge.
Written by our in-house scientists, articles cover every aspect of the baculovirus system – from its discovery and background, to practical applications in research and industry. We cover both the day-to-day challenges and more complex cases faced by ourselves and customers. You can also keep up to date on all the news, developments and promotional offers from the company.
If you have a question about any of the content featured, or simply want more information on a particular topic, then do not hesitate to get in touch with us directly at email@example.com. In addition, OET offers everything needed to aid your own insect virus research. As creators of the world renowned flashBACTM technology, our range of baculovirus expression vectors are designed for simple and rapid recombinant protein production. This is supported by an optimised range of transfer plasmids, transfection reagents, and cell culture media to ensure a powerful and versatile system. Browse the full collection of products at https://oetltd.com/shop/
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A question that often comes up from clients is what is the best signal peptide to use for baculovirus-expressed secreted recombinant proteins? While we have discussed the best flashBAC™ variant to use for the production of secreted proteins in a previous blog, we have never considered the role of the signal peptide. (more…)