Frequently Asked Questions:

flashBAC™ Baculovirus Expression Systems

flashBAC™ Baculovirus Expression Systems are a streamlined platform for the production of recombinant baculoviruses. Genetic optimization of the Autographa californica nucleopolyhedrovirus (AcMNPV) genome yields recombinant virus in a single step and eliminates the need for separation of recombinant and parental virus by plaque purification. flashBAC™ systems and pOET Insect and BacMam Transfer Plasmids are optimized for use with TransIT®-Insect Transfection Reagent.

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in the flashBAC™ Baculovirus Expression Systems?
All flashBAC™ Baculovirus Expression Systems include flashBAC™ DNA and the pAcRP23-lacZ positive control plasmid. They are provided in either 5 or 24 reaction quantities.

Q2. How does the flashBAC™ Baculovirus Expression System work?
To generate recombinant baculovirus using the flashBAC™ System, insect cells are transfected with TransIT® -Insect Transfection Reagent, flashBAC™ DNA, and a transfer plasmid (e.g. pOET1, pOET1C_6xHIS, or pOET6) containing the gene of interest. Homologous recombination within insect cells inserts the gene of interest under the control of the polyhedrin (polh) promoter, and restores the function of ORF1629 allowing viral DNA to replicate and produce virus particles. The recombinant virus genome replicates to produce baculovirus that can be harvested directly from the culture medium of transfected insect cells.

Q3. Which of the three flashBAC™ Baculovirus Expression Systems should I choose for expressing my gene of interest?
The right flashBAC™ System for expressing your gene of interest will depend on the characteristics of the protein being expressed and the overall goal of the experiment. For general guidelines, please refer to the following:

flashBAC™: With the original flashBAC™ expression vector, protein expression and secretion is improved by deletion of the chitinase gene from the Autographa californica nucleopolyhedrovirus (AcMNPV) genome. This vector is suitable for expression of most simple nuclear or cytoplasmic recombinant proteins.
flashBAC™ GOLD: flashBAC™ GOLD builds upon the basic flashBAC™ technology with the deletion of the chitinase and cathepsin protease genes from the AcMNPV genome. This system is ideal for high level expression of membrane and secreted proteins as well as proteins that are more susceptible to degradation.
flashBAC™ ULTRA: flashBAC™ ULTRA is the most advanced flashBAC™ vector system and is optimized by deletions in the chitinase, cathepsin, p10, p74 and p26 genes. flashBAC™ ULTRA is the ideal system for the most difficult to express recombinant proteins including nuclear, cytoplasmic, membrane and secreted proteins.

Q4. Can the pAcRP23-lacZ positive control vector be used as a cloning vector?
The pAcRP23-lacZ contains a lacZ gene under control of the polyhedrin (polh) promoter and does not contain a multiple cloning site. The control vector is meant to confirm recombination within insect cells and is not recommended for subcloning.

Q5. What is the size of the flashBAC™ DNA?
The flashBAC™ Baculovirus Expression System is based on a modified AcMNPV genome and is approximately 136 kb in size.

Q6. Which transfer vectors are compatible with flashBAC™ Expression System DNA?
The flashBAC™ system is compatible with all baculovirus transfer vectors that are based on homologous recombination in insect cells at the polyhedrin gene locus. Compatible transfer plasmids available through Mirus Bio LLC are as follows:

pOET1 (MIR 6150) and pOET1C_6xHis (MIR 6151) for high titer baculovirus production
pOET6 BacMam (MIR 6152) for high titer BacMam virus production

Other compatible examples include pBacPAK8/9, pAcUW31 and pBacPAK-His1/2/3 (BD Biosciences Clontech). pFastBac vectors, which are designed for site-specific transposition in E. coli using the Bac-to-Bac system (Life Technologies) are NOT compatible. A list of compatible plasmids sold through external sources can be found here.

Q7. What is a BacMam vector? What cells can I use with the BacMam virus?
A BacMam vector is a baculovirus transfer vector designed to facilitate the expression of foreign genes in mammalian cells under the cytomegalovirus (CMV) immediate early gene promoter. The purified baculovirus generated with insect cells can be used to transduce most mammalian cell types in a titer-dependent manner.

Q8. What transfection reagent is recommended for use with flashBAC™ Baculovirus Expression System?
We recommend TransIT®-Insect Transfection Reagent with the flashBAC™ Expression Systems. TransIT®-Insect Transfection Reagent is a proprietary, non-liposomal, animal-origin-free formulation developed for high transfection performance in insect cell lines.

Q9. What is the mechanism of action of TransIT®-Insect Transfection Reagent?
The TransIT®-Insect Reagent complexes with negatively charged DNA to form net positive lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis.

Q10. Which insect cell lines are suitable for the flashBAC™ Baculovirus Expression System?
Sf9 or Sf21 cells are generally used for co-transfections, virus amplification and plaque assays. T. ni cells are often used to maximize protein yields but are not recommended for virus production and amplification.

Q11. Do I need to perform a plaque assay before I begin protein production experiments?
Plaque purification to separate parental from recombinant virus is not necessary with the flashBAC™ Expression System. For optimal protein yield, it is recommended that baculovirus titers be determined via plaque assay or a comparable method prior to infection of insect cells.

Q12. How do the flashBAC™ Baculovirus Expression Systems compare to other commercially available baculovirus expression systems?
The flashBAC™ Expression System is the simplest and least time-consuming baculovirus expression system available that yields 100% recombinant virus in a single step. For direct competitor comparisons, please see the flashBAC™ Baculovirus Expression System product page.

Q13. What titers can I expect using the flashBAC™ Expression System?
Baculovirus titers will vary based on the properties of the gene of interest cloned into the transfer vector. A titer of approximately 1 x 10⁷ pfu/ml at day 5 post-transfection is expected with the pAcRP23-lacZ control plasmid, and titers of ≥ 1 x 10⁸ are typically observed after P1 amplification.

Q14. How should the flashBAC™ Expression System components be stored?
Store the components as follows:

Store flashBAC™ DNA at 4°C. DO NOT FREEZE.
Store Control Transfer Plasmid (pAcRP23-lacz) at -20°C.

PROTOCOL QUESTIONS AND ANSWERS

Q15. Which serum-free medium should I use for preparing flashBAC™ DNA + transfer vector DNA + TransIT®-Insect transfection complexes?
Always prepare TransIT®-Insect Reagent:DNA complexes in serum-free growth medium. Mirus recommends Grace’s Insect Basal Medium.

Q16. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q17. Is a media change required post-transfection?
No, a media change is not required following transfection. However, media will need to be added back into transfected wells 5-24 hours post-transfection. For further details, see section I-D of the full protocol.

Q18. What incubation conditions should be used for baculovirus production?
The optimal temperature for Sf9 and Sf21 growth and infection is 27-29°C. If the insect culture medium used employs a phosphate buffering system (e.g. Grace’s, SF-900), a CO₂ incubator is not required. Use vented shake, spinner, or tissue culture flask caps to ensure adequate dissolved oxygen content.

Q19. How can I validate delivery of the flashBAC™ Baculovirus Expression System?
To verify efficient transfection, use TransIT®-Insect Transfection Reagent to deliver the positive control plasmid provided in the flashBAC™ kit (pAcRP23-lacZ). LacZ-positive virus plaques can be stained by following the plaque assay procedure outlined in Section III of the flashBAC™ Baculovirus Expression Systems full protocol using X-gal rather than Neutral Red.

Q20. How long can I store my baculovirus stocks?
Baculovirus stocks may be stored for up to 12 months at 4°C, though loss of titer can occur during this time. To minimize titer loss, add 2-5% serum. If the baculovirus stock is stored for greater than 3 months, titer the virus before use and re-amplify if necessary. For long-term storage, virus should be aliquoted and stored at –80°C. Due to reduction of virus titer by freezing, avoid multiple freeze thaws and re-amplify virus before use. Storage at –20°C or in liquid nitrogen is not recommended.

TROUBLESHOOTING QUESTIONS

Q21. What can I do to further improve my baculovirus amplification titers with the flashBAC™ Baculovirus Expression System?
Multiple factors can contribute to transfection efficiency and ultimate baculovirus titer; refer to the following critical troubleshooting tips to improve your transfection results:

Cell density (% confluence) not optimal at time of transfection - See the full protocol for recommended plating and amplification recommendations. Baculovirus replication is inhibited when cells are seeded too densely. Depending on the cell type, higher or lower densities may be required for optimal amplification.
Cells not actively dividing at the time of transfection - Maintain insect cells used for the production of recombinant viruses in log-phase growth and discard after passage 30. Subculture cells before they become overgrown and enter stationary phase, generally every 3-4 days. Cells used for transfection and amplification should be in log-phase growth with a viability of >95%.
Suboptimal P0 volume used for amplification - The virus titer from the co-transfection may be too low for the volume of cells being infected. To determine the optimal harvest time, leave the infection for longer than 5 days and monitor virus amplification using a phase-contrast microscope. Conversely, viral titers will be lower if too much virus was added to the culture of cells and only one round of amplification occurred.
Poor quality of P0 virus stock - If the P0 stock has been stored for longer than 3 months, you may need to re-titer. If the titer has decreased, increase the volume of P0 used for amplification.
Culture time post-transfection - Determine the optimal transfection incubation time for each cell type and experiment. Test a range of incubation times. For P0 and P1 baculovirus production, the best incubation time is generally 4-5 days.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Frequently Asked Questions:

flashBAC™ Baculovirus Expression Systems

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

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