flashBAC Manual - Oxford Expression Technologies

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8.2 Amplification of recombinant <strong>flashBAC</strong> virusThe recombinant virus produced in 8.1 must be amplified forexperimental work. We strongly recommend amplifying virus incells grown in suspension culture, and the following gives aprotocol for amplifying 100-200 ml virus using the seed stock ofvirus harvested from the co-transfection as inoculum. Virusmay also be amplified in monolayer cultures and someguidance on this is given in the notes at the end of this protocol.The following procedures must be carried out using aseptictechnique.Required:• Seed stock of recombinant virus prepared in Protocol8.1.• 100-200 ml culture of log-phase insect cells (Sf21 orSf9) in appropriate medium [see section 10 for moredetails].• Shake culture flask (e.g., 1L sterile glass flask ordisposable Erlenmeyer flask) or spinner flask (e.g. 1LBellco Glass spinner flask) [see section 10 for moredetails].• Incubator set at 28°C.• Inverted phase-contrast microscope.• Sterile pipettes.• Disinfectant for discard.OXFORD EXPRESSION TECHNOLOGIES 26
Procedure:1 Prepare a 100-200 ml culture of Sf9 or Sf21 cells at anappropriate cell density (in log growth phase). SeeFigure 4.• The cell density will vary with the cell type and themethod of culture. As a guide, use Sf9/Sf21 cells inshake culture, in serum-free medium, at 2 x 10 6cells/ml or Sf21 cells, in serum-supplementedmedium, in spinner culture at 0.5 x 10 6 cells/ml. Seesection 10 for more information.• It is important that the cells are healthy and in logphase of growth to ensure that virus replicationoccurs efficiently to amplify high titre stocks of virusfor subsequent use in expression studies. Youshould check the cell density and cell viability ofyour culture before using it to amplify virus (seesection 10 for details).• It is also vitally important that the cells are infected ata very low multiplicity of infection (moi) (< 1 pfu/cell).This means that initially few cells are infected; thevirus replicates to release BV, which then infectsmore cells and so on. In this way multiple rounds ofreplication occur and high virus titres are obtained; italso reduces the chances of any defective virusparticles occurring. If the cells are infected at highmoi (>1 pfu/cell), all the cells will be infected initiallyand only one round of replication will occur, giving apoor virus amplification and low titre virus stock.OXFORD EXPRESSION TECHNOLOGIES 27
Page 1 and 2: User GuideJanuary 2008OXFORD EXPRES
Page 3 and 4: flashBAC User GuideContentsPage1 Li
Page 5 and 6: 1 Limited Use Licence1 In this Lice
Page 7 and 8: 2 Kit ContentsAll reagents and mate
Page 9 and 10: 6 Safety Requirements1 These resear
Page 11 and 12: Figure 1. A rod-shaped baculovirus
Page 13 and 14: development of the baculovirus expr
Page 15 and 16: the gene of interest, under the con
Page 17 and 18: ie1 etc. Examples include pBacPAK8/
Page 19 and 20: OXFORD EXPRESSION TECHNOLOGIES 19
Page 21 and 22: • Sterile baculovirus transfer ve
Page 23 and 24: 6 Just before the end of the incuba
Page 25: affected by using semi-pure transfe
Page 29 and 30: cycles. Upon freezing, the viral ti
Page 31 and 32: 8.3 Plaque assay to titre recombina
Page 33 and 34: convenient to prepare 0.5 ml diluti
Page 35 and 36: from each of the 35 mm dishes, by t
Page 37 and 38: plates give a titre of:25 x 10 6 x
Page 39 and 40: OXFORD EXPRESSION TECHNOLOGIES 39
Page 41 and 42: 9 Analysis of gene expression: a gu
Page 43 and 44: for 1 hour, remove the inoculum and
Page 45 and 46: Multiplicity of infectionIn order t
Page 47 and 48: 10 A Guide to Insect Cell CultureIt
Page 49 and 50: orbital shaker platform, whilst cel
Page 51 and 52: counting chamber (Figure 5), using
Page 53 and 54: Passaging suspension cultures of ce
Page 55 and 56: Table 3. Seeding densities of insec
Page 57 and 58: 11 Troubleshooting & FAQQ) Why are
Page 59 and 60: culture dishes within 1 h after pla
Page 61 and 62: Is the coding region of the gene do
Page 63 and 64: 11. Kitts P. A., Ayres M. D., Posse

flashBAC Manual - Oxford Expression Technologies

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