How to produce baculovirus?
How to produce baculovirus?
Baculovirus is produced by transfected Sf9 cells and secreted into the culture medium which subsequently infects the untransfected cells. The entire cell population becomes infected with baculovirus in 5 days and shows signs of viral infection, also called cytopathic effect.
Why use baculovirus expression system?
The baculovirus expression system is commonly used to generate recombinant proteins in insect cells at high production levels. Recombinant baculoviruses can accommodate large segments of foreign DNA.
What is Bacmid DNA?
The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase.
Are Sf9 cells adherent?
The images below show the morphology of healthy Sf9 insect cells in suspension and adherent cultures. Sf9 cells attach firmly to surfaces, and their small, regular size makes them exceptional for the formation of monolayers and plaques.
How do you transfect Sf9 cells?
Warm Transfection Medium to room temperature 2. Transfer 2.5mL of Sf9 at 1 x 10 cells/mL into each 24 deep well block. Add 3 uL FuGENE HD directly to the Transfection Medium, avoid touching the side of the tube when adding FuGENE. Incubate at room temperature for 5 minutes.
What type of virus is the baculovirus expression system?
In the laboratory and for manufacturing purposes, the most commonly used baculovirus is Autographa californica multiple‐capsid nuclear polyhedrosis virus (AcMNPV), a virus with a double‐stranded DNA genome of approximately 134 kb [10].
Do Sf9 cells require serum?
Sf9, Sf21, and High Five™ cells can be grown in serum-required or serum-free media (see Media Considerations, page 8, for more information).
How do you store Sf9 cells?
Store frozen Sf9 cells in liquid nitrogen (vapor phase) until ready to use. Frozen cells are supplied in and may be thawed directly into Sf-900 II SFM. Use the following procedure to thaw cells. Note: We recommend thawing Sf9 cells into suspension culture in shake flasks.
What kind of DNA is used in Sf9 cells?
Sf9 cells are cotransfected with the pVL-1392 plasmid and linearized baculovirus DNA to achieve homologous recombination of both components and production of functional baculoviruses encoding the protein of interest. Here, we provide a simple protocol for baculovirus generation using the BaculoGold kit, which works reliably in most cases.
How to establish a SF9 or Sf21 cell line?
1. Establish the cell line, Sf9 or Sf21, in Transfection medium (Expression systems #95-020). Cotransform the transfer vector (e.g., pBAC) with Saffire™ viral DNA (Orbigen# BVD-10001) into insect cells using FuGENE HD Transfection Reagent (Roche Applied Science #04709705001).
What happens when a Sf9 cell line is contaminated?
Accidental baculoviral contamination of an Sf9 cell culture can be easily recognized by typical signs of infection, for example, deformation of cell shape, reduced growth, and finally the death of the whole cell population. For baculovirus generation, in our hands the BaculoGold™ transfection kit from Invitrogen yielded the best results.
How often should a Sf9 cell line be seeded?
Normally, the volume of a suspension culture in shaker flasks should not exceed 40% of the flask volume. This ensures optimal mixing of the medium and oxygenation. Sf9 cell suspension cultures should be passaged three times weekly and seeded at a density of ~ 1 × 10 6 cells/mL.