flashBAC™ User Guide 2015 - Oxford Expression Technologies
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1. 8 Page Oxford Expression Technologies flashBAC User Guide 2015 Expression of foreign genes in insect cells using recombinant baculoviruses has become one of the most widely used eukaryotic expression systems The BEVS as it is called has several advantages over other expression systems e Safe to use baculoviruses only infect insects and polyhedrin negative viruses cannot survive in the environment e Can accommodate large genes or multiple genes as the rod shaped nucleocapsid can increase in length e Wide variety of promoters can be used not just polyhedrin to control level of expression and or temporal aspects of expression e Proteins made are usually functional and are cleaved processed correctly e Can be used to transduce mammalian cells and achieve gene expression by replacing polyhedrin gene promoter with a mammalian specific promoter e Insect cells are easy to grow and scale up at lower temperatures than mammalian cells and without the need for CO incubators However the BEVS is not without its disadvantages and these lie mainly in the labour intensive and technically demanding steps needed to produce and isolate recombinant viruses and the fact that glycosylation differs from mammalian cells the latter often has no effect on function but is important in considering therapeutic proteins The following section outlines the development of the BEVS over time and the fine tuning that has been achieved to improve2. It is convenient to monitor gene expression by setting up small scale monolayer cultures in either 35mm dishes or the wells of a 12 well plate Set up monolayers in dishes wells as described for co transfections plaque assays see 8 2 8 4 and leave the cells to recover for an hour Always take cells from log phase cultures to ensure that virus can infect the cells and replicate otherwise the polyhedrin gene promoter or other virus promoter will not be turned on and expression levels will be very low Infect 35 mm dishes with 200 ul virus inoculum or 12 well plate wells with 100ul Simply remove the medium add the inoculum drop wise and gently to the centre of the dish and leave to adsorb for 45 60 min with occasional rocking of the dishes Then replace the growth medium 2 ml for 35 mm dishes and 1 ml for 12 well plates Incubate at 28 C Always include a negative control mock infected cells for comparison If you have a known recombinant baculovirus you can add a positive control If you purchased a flashBAC kit you could make a recombinant virus with the control lacZ transfer vector and use this to set control infections to look for beta galactosidase production see Figure 3 25 Page Oxford Expression Technologies flashBAC User Guide 2015 We normally test expression by harvesting the cells and or culture medium as needed at 72 hpi initially If you want to test the culture medium for secreted protein harvest the medium c
3. OXFORD EXPRESSION TECHNOLOGIES A guide to making recombinant baculoviruses using BacPAK6 or flashBAC User Guide 2015 Oxford Expression Technologies flashBAC User Guide 2015 Contents Oon A UU N FB 10 Limited Use License flashBAC amp BacBAK 6 Kit Contents Essential Information and Technical Assistance Safety Requirements flashBAC amp BacPAK6 kit and related products ordering information Introduction to the Baculovirus Expression Vector System Introduction to the BacPAK6 and flashBAC systems Making recombinant baculoviruses Insertion of gene into and choice of transfer plasmid Co transfection of insect cells with flashBAC or BacPAK6 DNA and transfer plasmid DNA to make recombinant virus Plaque purification of recombinant BacPAK6 virus Amplification of recombinant baculoviruses Titration of recombinant baculoviruses Making recombinant viruses in 24 well plate format Analysis and optimisation of gene expression Trouble Shooting and FAQ References 14 17 20 22 24 25 26 28 2 Page Oxford Expression Technologies flashBAC User Guide 2015 10 11 12 13 14 15 Limited Use Licence for flashBAC virus DNA In the License the following expressions shall have the following meanings DNA shall mean deoxyribonucleic acid Fee shall mean the fee invoiced for the Materials by the Licensor to the Licensee Licensee shall mean the purchaser of the Materials Licens
4. High MOI will lead to lower titres and very low MOI will work but you may need to leave the cells longer to achieve high titres Did he cells look infected grainy and swollen nuclei under the microscope Could the foreign gene product be affecting budded virus production Q Why don t see plaques in my plaque assay Were the cells in good condition see Q on cells above Double check the cell density that was plated too high and cells cannot undergo enough rounds of replication to form a plaque they will be like pin pricks and hard to see too low and the cells do not close up to form a monolayer so edges of plaques can be ragged and hard to spot Are there any cells left at all look under microscope if the dishes dried out at any time there will be no cells left and no plaques Was the agarose overlay too hot which may have killed the cells Did you remember the liquid overlay with 10 for TC100 Was the virus titre too low to be detected 0 try again with lower dilutions or even neat virus plated out Or was titre too high and you need to plate out higher dilutions to see plaques they may have merged together and be hard to see did you change tips between dilutions to avoid carry over Was the Neutral red freshly diluted ready for use Q My plaque assay overlay has cracks or fell out when inverting or the plaques are smeared or only around the edges of the dish A If the virus inoculum is not removed before add
5. gal You need to pick 3 6 plaques for each virus Select well isolated plaques from a dish where there are no blue plaques see Figure 7 If the dilutions were unsuitable i e too few or to many plaques per dish you may have to redo the plaque assay adjusting the dilutions to obtain dishes with well isolated plaques and no blue plaques With experience you can cut down the range of dilutions plated once you know the general titre of virus that you obtain from a co transfection We recommend starting with a wide range as transfection efficiency varies considerably Figure 7 Plaque assay in Sf21 cells stained with Neutral red to show well isolated plaques a crowded plaques b and merged plaques c 19 Page Oxford Expression Technologies flashBAC User Guide 2015 14 To pick a plaque you need to take up a plug of agarose from the centre of a plaque using a Pasteur pipette or Gilson tip Disperse the plug of agarose into 500 ul growth medium in a micro centrifuge tube and vortex to release the virus from the agarose into the medium Store in the dark at 4 C 15 Amplify the plaque picked virus by inoculating either a 35mm dish or a T25 flask of Sf21 or Sf9 cells using 100 ul 35 mm dish or 250 ul T25 flask of your 500 ul as inoculum To do this seed a 35 mm dish or T25 flask with cells to form a sub confluent monolayer and after an hour or so remove the medium and replace with the inoculum for 45 60 mins Then add 2 ml
6. just add 0 1 0 2ml fresh medium to each plate to prevent drying of cells Prepare more agarose overlay medium and carry on but don t forget to remove the extra medium you added to each dish Allow the agarose overlay to set at room temperature Then add a 1 ml liquid overlay of growth medium to feed the cells and prevent them from drying out Place the dishes in a plastic box and incubate at 28 C for 3 4 days Three days for Sf21 and four days for Sf9 cells Add 1 ml growth medium containing 15 ul 2 v v X gal in DMF to each dish to stain for blue parental plaques Incubate at 28 C for 5 6 h Conveniently this is done in the course of a normal working day Blue plaques should start to develop during this time Prepare the Neutral Red stain in water to 5 mg ml d water and filter sterilize or purchase ready made from Sigma for example Dilute 1 in 20 with sterile PBS for use Different batches of Neutral Red may differ in their efficacy Sometimes 1 in 40 dilutions give better results Do not store diluted stain it will form a precipitate The concentrated stock is stable at room temperature for several months if sterile Add 1 ml diluted neutral red stain to each dish Do not remove the X gal already added Incubate at 28 C for 16 hours overnight Decant all liquid and view plaques on a light box Recombinant virus plaques will appear clear in a sea of red healthy cells Parental non recombinant plaques will stain blue with X
7. medium you added to each dish Allow the agarose overlay to set at room temperature Then add a 0 5 ml liquid overlay of growth medium to feed the cells and prevent them from drying out Place the dishes in a plastic box and incubate at 28 C for 3 4 days Three days for Sf21 and 4 days for Sf9 cells Prepare the stain by dissolving Neutral Red in water to 5 mg ml d water and filter sterilize or purchase ready made from Sigma for example Dilute 1 in 20 with sterile PBS for use Note some batches of Neutral Red may work better at 1 in 40 dilution do not store diluted stain as it precipitates Add 0 5 ml diluted neutral red stain to each dish and incubate for 3 4 hours Decant the remaining liquid and view plaques on a light box Recombinant virus plaques will appear clear in a sea of red healthy cells It sometimes takes a few hours for plaques to be really visible Count the plaques from wells where there are a countable number of plaques 10 20 Average the plaque count from the triplicate dishes and note the dilution that gave rise to these plaques Determine the virus titre as follows Average number plaques x dilution factor x 10 plaques ml in the original virus stock Inverse of dilution because only 0 1 ml was added to dish For example if the average number of plaques was 15 taken from the 10 dilution wells the virus titre would be 15x10 x 10 1 5 x 10 pfu ml Note that virus titres will drop after stora
8. must be carried out using aseptic technique as the DNA complexes will be introduced in insect cells in the absence of antibiotics Read through the whole protocol before starting to check you have all the reagents and equipment needed Check safety advice and MSDS data sheets where appropriate We recommend wearing PPE such as lab coats and gloves at all times Provided in the kit flashBAC DNA any type or BacPAK6 DNA use 100 ng 5ul DNA per co transfection Positive control transfer plasmid DNA expressing lacZ use 500 ng 5ul per co transfection flashBAC kit only Also needed 12 well plate or 35mm tissue culture dish seeded with a sub confluent monolayer of Sf21 or Sf9 cells one dish well for each co transfection and one for a control See OET s Cell Culture Manual for details on insect cell culture it is vital for transfection success that cells used are taken from a culture that is in log phase growth virus can only replicate when cells are in log phase A sub confluent monolayer is one in which there are spaces around each cell so there is room for each cell to divide in the 24 hours after co transfection Serum free insect cell culture medium we recommend using TC100 medium as a transfection medium but most serum free medium will also work Growth medium serum free or TC100 with 10 serum as preferred Sterile transfer plasmid DNA containing gene to be expressed see 8 1 for details 500 ng per tr
9. or 12 well plates one dish well per condition or set up small 20 ml shake cultures and take samples 2 ml at various time points The latter is better if you are planning on scaling up in future You may also need to do pilot protein purification and small scale shake cultures can work well for this too Always do control mock infected dishes or take samples prior to infecting shake cultures Parameters to optimise include e Multiplicity of infection start by comparing 1 3 and 10 pfu cell e Cell line Sf9 Sf21 TniHi5 SuperSf9 cells e Time to harvest 0 24 38 72 96 hpi e flashBAC variant see introduction 8 4 Scaling up production There are many ways to scale up insect cell culture and hence virus or protein production The simplest is to use large scale shake flasks In this way up to 1 25 L cells can be infected at one time The key to success is to ensure that flasks are not overfilled aim for maximum surface area and that cells are shaken at a high rpm to ensure good aeration GE Healthcare s wavebags are also relatively easy to use but are expensive and require access to a Wave Bioreactor The OET Cell Culture Handbook has more information on this topic 9 Trouble shooting and FAQ Q Why are my cells not growing well A The most likely problem with cells occurs when they have been allowed to reach stationary phase before passaging If this stress happens to a culture 2 or 3 times then the cells no
10. to semi quantify expression levels es i ua In most cases FBG improves secretion aS UMAR ABD levels of proteins a a FB1 FBG1 FB2 FBG2 FB3 FBG3 FB1 FBG1 FB2 FBG2 FB3 FBG3 wi FB4 FBG4 FB5 FBG5 FB6 FBG6 FB4 FBG4 FB5 FBGS FB6 FBG6 12 Page Oxford Expression Technologies flashBAC User Guide 2015 7 A laboratory guide to making recombinant baculoviruses using either BacPAK6 or flashBAC 7 1 Choice of transfer plasmid Both the BacPAK6 and flashBAC systems use transfer plasmids to mediate transfer of the gene s to be expressed into the virus genome at the polh locus A large number of transfer plasmids are available from OET Ltd and other suppliers X Y Z Please see the OET website for more details or consult the following review articles Transfer plasmids can be grouped as follows Polyhedrin gene promoter P10 gene promoter Dual promoters Multiple promoters BacMAM plasmid promoters Late gene promoters Purification tags Fusion vectors Signal peptides Start codons Codon optimisation Membrane anchors Simple vector such as pOET1 or 2 that has a multiple cloning site MCS to insert your gene under control of the strong very late polyhedrin gene promoter Another strong very late gene promoter For dual expression of genes usually one under polyhedrin and one under P10 gene promoters such as pOET5 A mix of copes of polyhedrin and P10 promoters Caref
11. well plate seal and store at 4 C in the dark To amplify virus follow protocol 7 4 as a guide use 250 ul to infect 50 100 ml of Sf9 cells 8 Analysis and Optimisation of Gene Expression This section provides a guide to the analysis of gene expression from recombinant virus made using either the BacPAK6 or flashBAC systems It is not intended to be prescriptive simply a guide to help you get started 8 1 Quick check for gene expression After the co transfection or after amplification of PO virus to give P1 remaining cells in the monolayer can be harvested and used to test for gene expression by SDS PAGE and or Western blotting However the expression levels are variable at these stages so many people prefer to wait until they have a high titre stock of virus P1 or P2 Some of the expression after the co transfection will also be transient expression from the transfer plasmid itself 8 2 Test expression by infecting cells with high titre virus stocks It is always best to test expression using a virus with a known titre That way you can control the multiplicity of infection Normally the best levels of expression are obtained with high MOls 5 10 pfu cell so that all the cells are infected simultaneously and a synchronous infection is established However for a few proteins best expression is obtained at lower MOI We therefore recommend that expression testing includes a range of MOI 0 5 2 5 and Sisa good starting point
12. 2 100710 100711 100712 100713 100714 100715 100716 100717 Catalogue number 600100 600105 600102 600103 600104 Catalogue Number 500200 500300 500301 500302 500303 500304 500305 500306 500307 500308 500309 500310 6 Page Oxford Expression Technologies flashBAC User Guide 2015 6 Introduction to the Baculovirus Expression System and flashBAC BacPAK6 technology 6 1 Baculoviruses Baculoviruses are insect viruses predominantly infecting insect larvae of the order Lepidoptera butterflies and moths A baculovirus expression vector is a recombinant baculovirus that has been genetically modified to contain a foreign gene of interest which can then be expressed in insect cells under control of a baculovirus gene promoter The most commonly used baculovirus for foreign gene expression is Autographa californica nucleopolyhedrovirus AcMNPV AcMNPV has a circular double stranded super coiled DNA genome 133894 bp Accession NC_001623 packaged in a rod shaped nucleocapsid The nucleocapsid can be extended lengthways and thus the DNA genome can accommodate quite large insertions of DNA The ACMNPV genome forms the basis of the flashBAC or BacPAK6 DNA provided in this kit AcMNPV has a bi phasic life cycle Figure 1 resulting in the production of two virus phenotypes budded virus BV and occlusion derived virus ODV BV contain single rod shaped nucleocapsids enclosed by an envelope derived fro
13. 9 is restored and the recircularised DNA can replicate to produce recombinant budded virus This reduced even further the chance of parental virus replicating and resulted in an increase in the recovery of recombinant virus to more than 90 It also introduced a useful blue white selection system with non recombinant virus giving rise to blue plaques and recombinant virus to white plaques It was thus easier to achieve purified virus with a single round of plaque purification It is not 100 because it is impossible to ensure that every molecule of DNA is triple digested and any circular DNA remaining can replicate and produce non recombinant virus The triple cut linear BacPAK6 virus DNA is available from OET see page 5 We are also pleased to offer BacPAK6 Sec which has a deletion in the chitinase gene to aid expression of membrane targeted and secreted proteins Practical techniques to make recombinant BacPAK6 viruses are included in this User Guide Despite this fine tuning and optimisation of the system a number of steps are still required to make recombinant baculoviruses thus making it more time consuming than bacterial expression systems and less amenable to scale up and high throughput automation 6 4 The flashBAC system The flashBAC system is a new platform technology for the production and isolation of recombinant baculoviruses Importantly flashBAC has been designed to remove the need for separation of recombin
14. 99 1513 Possee R D amp Howard S C 1987 Nuc Acids Res 15 10233 10248 Kitts P A Ayres M D amp Possee R D 1990 Nuc Acids Res 11 5667 5672 Kitts P A amp Possee R D 1993 Biotechniques 14 810 817 Patent applications EP1144666 W00112829 amp AU6460800 Luckow V A Lee S C Barry G F amp Olins P O 1993 J Virol 67 4566 4579 Hawtin R E et al 1995 Virology 212 673 685 Hawtin R E et al 1997 Virology 238 243 253 Thomas C A et al 1998 J Virol 72 10207 10212 Saville G P Patmanidi A L Possee R D amp King L A 2004 J Gen Virol 85 821 831 Possee R D Saville G P Thomas C J Patminidi A amp King L A 2001 In Prospects for the development of insect factories Proceedings of a Joint International Symposium of Insect COE Research Program and Insect Factory Research Project October 22 23 Tsukuba Japan Vaughn J L Goodwin R H Tompkins G J amp McCawley P 1977 In Vitro 13 213 217 Hink W F 1970 Nature 226 466 467 Hink W F amp Vail P V 1973 J Invertebr Pathol 22 168 174 28 Page
15. add an equal volume of pre warmed growth medium 28 C Keep warm to prevent setting we use a temporary clean water bath comprising a beaker of hot tap water You need 1 ml final overlay per dish If the agarose in water sets it is easy to melt again by boiling If the agarose overlay with growth medium sets you cannot re melt You have to start again We often prepare several small batches of agarose in water and let them set and then melt each aliquot as we need it 7 ml is convenient 23 Page Oxford Expression Technologies flashBAC User Guide 2015 10 11 12 13 14 8 6 At the end of the incubation period 4 remove the inoculum using a pipette and discard into Virkon or other disinfectant Working quickly add 1 ml warm overlay to each dish allowing the agarose to flow down the side of the dish and spread slowly over the monolayer of cells Do NOT pipette into the centre of the dish Process one set of dishes per virus sample at a time If working in a hood keep the agarose overlay in a beaker or sandwich box filled with warm water to delay solidification If the agarose sets prematurely you can leave the dishes with virus inoculums for longer than 60 min without adverse effects If you have removed the virus and then find that your overlay medium has set just add 0 1 0 2ml fresh medium to each plate to prevent drying of cells Prepare more agarose overlay medium and carry on but don t forget to remove the extra
16. ansfection 14 Page Oxford Expression Technologies flashBAC User Guide 2015 Transfection reagent such as OET s baculoFECTIN II or flashFECTIN volume as indicated by the manufacturer Incubator set at 28 C 1 Virkon Amtec or other suitable disinfectant Inverted phase contrast microscope Plastic box to house dishes in the incubator Sterile pipettes and bijoux or other polystyrene containers to make up the transfection mix do not use micro centrifuge tubes made of polypropylene Method 1 For each co transfection you require one 35mm dish or one well of a 12 well plate containing sub confluent Sf9 or Sf21 cells If you are making a virus with the control vector provided in the flashBAC kit add an extra dish well of cells It is also good practice to have one dish well for a mock transfection in which no DNA is added Do not use TniHi5 cells to make viruses as they are prone to mutations that affect gene expression Seed the dishes wells with cells at least one hour before use to allow cells to attach and recover Ensure cells were taken from a log phase culture of cells that were at least 90 viable As a rough guide you need about 1 5 x 10 Sf21 or Sf9 cells per 35mm dish to form a sub confluent monolayer For 12 well plates add 0 4 x 10 Sf9 Sf21 cells per well The volume of medium should be 2 ml in 35mm dishes and 1 ml in 12 well plates Ensure cells are evenly distributed over the surface of the dish well 2 Durin
17. ansfection mix L l Tronstect E S Figure 4 Overview of how the flashBAC harvest after 5 days system works in practice The co transfection fe mix comprises flashBAC DNA transfer c plasmid with gene to be expressed and i transfection reagent Up to 24 viruses can be made at one time using a 24 well plate dish either manually or using a simple liquid handing robotic platform 11 Page Oxford Expression Technologies flashBAC User Guide 2015 Figure 5 Production and analysis Production of secreted recombinant proteins from Sf9 cells 72 hr post infection using seed stock viruses aE recombinant proteins using flashBAC viruses P1 stock in Sf9 cells and probed with anti His antisera Thanks to Dr Ray Owens 80 Oxford Protein Production Facility for beta testing flashBAC number of secreted 10 ul media analysed on 10 20 SDS PAGE W blot with anti His Mab 12 3 4 5 6 78 9 10 11 12 13 14 15 16 17 18 19 20 21 kB 1 OPPF 1904 OPPF 1938 15 OPPF 1906 2 OPPF 1906 OPPF 1942 16 OPPF 1916 3 OPPF 1910 10 OPPF 2045 17 OPPF 1930 4 OPPF 1916 11 OPPF 2047 18 OPPF 1938 5 OPPF 1924 12 OPPF 2049 19 OPPF 1942 6 OPPF 1930 13 OPPF 2055 20 OPPF 2055 T OPPF 1936 14 OPPF 1904 21 Mock Secreted proteins flashBAC vs flashBACGOL2 Figure 6 Expression of secreted proteins 1 6 using flashBAC FB or flashBAC GOLD FBG Western blots probed with anti His antisera are shown as are densitometry results
18. ant virus from parental virus so no plaque purification steps are needed The production of recombinant virus has been simplified to a single stage procedure that is fully amenable to high throughput manipulations multiple recombinant viruses can be made at one time using 24 well plates either manually or using simple robotic systems The flashBAC technology builds on the BacPAK6 technology At the heart of the new system is an ACMNPV genome that lacks part of the essential gene ORF 1629 and contains a bacterial artificial chromosome BAC at the polh locus replacing the polh coding sequence The essential gene deletion prevents virus replication in insect cells and the BAC allows the virus genome to be maintained in bacterial cells as a bacmid Circular virus DNA is isolated from bacterial cells and purified ready for use in flashBAC kits and co transfections to make recombinant viruses A recombinant baculovirus is produced simply by co transfecting insect cells with flashBAC DNA and a transfer plasmid containing the gene to be expressed Figure 4 Homologous recombination within the insect cells 1 restores ORF 1629 allowing the recombinant virus to replicate 2 removes the BAC sequences and 3 inserts the foreign gene under control of the polh promoter or other promoter chosen that is in the transfer plasmid The recombinant virus budded virus is harvested from the co transfection medium and becomes the seed stock PO of recombina
19. ashBAC PRIME ation from OET Ltd Details 5 reactions 24 reactions 96 reactions Bulk 3 reactions 5 reactions 24 reactions 96 reactions Bulk 3 reactions 5 reactions 24 reactions 96 reactions Bulk 3 x 3 reactions 4 x 3 reactions 5 reactions 24 reactions Bulk BacPAK6 products available from OET Ltd BacPAK6 Linearised DNA BacPAK6 Linearised DNA BacPAK6 Linearised DNA BacPAK6 Sec Linearised DNA BacPAK6 Sec Linearised DNA BacPAK6 Sec Linearised DNA 5 reaction kit 24 reaction kit 96 reaction kit 5 reaction kit 24 reaction kit 96 reaction kit Related reagents available from OET Ltd Transfection reagents Product flashFECTIN flashFECTIN baculoFECTIN II baculoFECTIN II Transfer plasmids Product pOET1 10 pg pOET1N_6xHis 10 ug pOET1C_6xHis 10 ug pOET2 10 ug pOET2N C_6xHis 10 ug pOET2C_6xHis 10 ug Size 300 ul 1ml 150 ul 1ml Details Price 150 00 650 00 2 595 00 POA 225 00 350 00 950 00 3 095 00 POA 250 00 400 00 1 128 00 3 695 00 POA 650 00 825 00 350 00 950 00 POA 250 00 750 00 2 695 00 250 00 750 00 2 695 00 Price 55 00 150 00 75 00 350 00 Polyhedrin gene promoter with multiple cloning site MCS Polyhedrin gene promoter MCS with N terminal 6xHis tag Polyhedrin gene promoter MCS with C terminal 6xHis tag As pOET1 but with reversed MCS Polyhedrin gene promoter MCS with N and C termi
20. ay Super Sf91 Super Sf92 Super Sf93 Frozen gt 1 x 10 cells per ampoule 21 x 10 cells per ampoule 21 x 10 cells per ampoule 21 x 10 cells per ampoule gt 1 x 10 cells per ampoule 132 00 132 00 132 00 132 00 137 00 Price 5 100 reactions 895 00 100 reactions 365 00 5 100 reactions 463 50 24 100 reactions 913 50 5 100 reactions 643 50 24 100 reactions 1 183 50 5 100 reactions 688 50 24 100 reactions 1 344 00 5 100 reactions 643 50 24 100 reactions 1 183 50 Live culture Medium On request Serum free On request TC100 with 10 serum On request Serum free On request Serum free On request Serum free Insect cell culture media products available from OET Ltd Product BaculoGROW II ESF 921 ES Transfection medium ES production boost additive ESF 921 delta methionine ESF 921 delta amino acid deficient ES Custom media production Size 500 ml 1L 3x1L 5x1L 10x1L Bulk gt 10L 20 ml 100 ml 100 ml 1L 1L Min 20L Shipping conditions Room temperature Room temperature Room temperature Room temperature Room temperature Room temperature Room temperature Medium Serum free Serum free Protein free Serum free Animal free Serum free nutrient additive Serum free methionine free Serum and amino acid free Custom to order 200104 200105 200106 200107 200108 Catalogue number 400101 10060
21. dish or 5 ml T25 flask growth medium no need to remove the inoculum and incubate for 4 5 day at 28 C The cells should be well infected under the microscope at the end of the infection period 16 Harvest the 2 ml or 5 ml of medium containing your PO seed stock virus Store at 4 C in the dark Use this to amplify a P1 working stock of recombinant virus to test gene expression see 7 4 17 The cell monolayers from the dish or flask used to amplify virus can be harvested and used to test for gene expression or to isolate DNA to do a PCR to check that the gene has gone into the virus genome 7 4 Amplification of recombinant baculoviruses to produce high titre stocks of virus This is a generic method to amplify recombinant baculovirus from PO to P1 or P1 to P2 etc We do not recommend serial passage of the virus stock because mutations can and do arise These can sometimes lead to reduced expression levels or loss of the gene Good practice is to amplify a 50 to 200 ml P1 stock for initial test of gene expression and optimisation of expression Some of this virus should also be frozen down at 80 C for long term storage Do not store virus at 20 C Virus can be stored in the dark at 4 C for a few months but in the absence of serum the titre can start to drop after a few weeks We recommend adding serum to 5 for all viruses stored at 4 C If you cannot do this then freeze aliquots of P1 virus at 80 C after adding serum to 2 5 and use thes
22. e to establish new P2 stocks when needed If you are planning to scale up protein production beyond a few hundred mls you will need to produce some P2 virus to use for experiments even P3 Again you may need to think about storing some of this at 80 C Most recombinant baculoviruses will amplify to titres in the region of 1 2 x 10 pfu ml Sometimes a foreign protein inhibits affects budded virus formation or is toxic and virus titres will be lower Anything above 1 2 x 10 should be enough To have the best chance of producing a good high titre stock of P1 or P2 virus use Sf9 cells growing in shake cultures in serum free growth medium or Sf21 cells growing in TC100 with serum in stirred cultures See the OET Cell Culture Manual for more details on insect cell culture Whichever cells are used they must be harvested in log phase and be at least 90 viable when used to set up a new culture ready to infect This is because the virus needs cells in a dividing state to be able to replicate To avoid accumulating mutants always infect cultures at very low multiplicity of infection MOI and allow the virus to undergo multiple rounds of replication this also achieves the highest titres possible If you infect cells at high MOI all the cells in the culture will be infected at the start and the virus will undergo one round of multiplication with a higher chance of cross over or other mutation events occurring Read through the method before
23. efine We have also noted that plaque assays conducted with Sf9 cells and serum free medium produce plaques that quickly fade after staining with Neutral Red 2 Make 1 in 10 dilutions of your transfection virus stock from 1 in 10 10 to 1 in 10 10 Use 50 pl virus and 450 ul growth medium as diluent at each step Mix the virus and diluent between each step and change tip pipette each time to avoid carry over 3 Remove the medium from the dishes of cells using aseptic technique and add 100 ul of diluted virus drop wise to the centre of each dish Plate a range of dilutions and two plates per dilution the aim is to get well isolated plaques on at least one dilution We normally plate the 1 in 100 10 to 10 dilutions in duplicate dishes and use two dishes as mock infected controls use medium only It is important that the cell monolayers do not dry out during this process of virus inoculation Do not leave lids off dishes for long periods If working in a class 2 hood be aware the air flow can dry plates very quickly If after staining your monolayer appears with a shiny red patch devoid of cells you have allowed the monolayer to dry out 4 Allow the virus to adsorb and be taken up into the cells at room temperature for 45 60 min Rock the dishes every few minutes to ensure even coverage of the inoculum Do NOT put the cells in the incubator as they will dry out 5 During this time prepare the overlay Dissolve agarose in water
24. elop and are not quite so easy to define Make 1 in 10 dilutions of your virus stock from 1 in 10 10 to 1 in 10 107 Use 50 ul virus and 450 ul growth medium as diluent at each step Mix the virus and diluent between each step and change tip pipette each time to avoid carry over It is convenient to do this in a 12 well plate Remove the medium from the dishes of cells using aseptic technique and add 100 ul of diluted virus drop wise gently to the centre of each dish Plate a range of dilutions from 10 to 10 and three wells per dilution 12 wells The aim is get at least one set of wells with a countable number of plaques It is important that the cell monolayers do not dry out during this process of virus inoculation Do not leave lids off dishes for long periods If working in a class 2 hood be aware the air flow can dry plates very quickly If after staining your monolayer appears with a shiny red patch devoid of cells you have allowed the monolayer to dry out Allow the virus to adsorb and be taken up into the cells at room temperature for 45 60 mins Rock the dishes every few minutes to ensure even coverage of the inoculum Do NOT put the cells in the incubator as they will dry out During this time prepare the overlay Dissolve agarose in water to 2 w v by boiling water bath or microwave oven take appropriate safety precautions You need 0 5 ml per dish of cells Cool the overlay to hand hot about 50 55 C and
25. ene properly under control of the polyhedrin gene promoter is the first ATG the ATG of your gene If not you need to address the construct and make a new virus If you are using tags to detect the gene check they are in frame Did you try optimising expression see above In particular sometimes T ni cells yield protein when Sf9 do not Finally if you have exhausted all avenues there are a very very few genes that for unknown reasons do not express Most have been found to be toxic to the cell But check all of the above before thinking this 10 References 20 21 22 Van Regenmortal M H V et al 2000 Virus Taxonomy Classification and Nomenclature of Viruses Seventh Report of the International Committee on Taxonomy of Viruses San Diego Academic Press Vail P V Jay D L amp Hunter D K In Proc IVth Int Collog Insect Pathology 297 304 College Park MD 1971 Smith G E Summers M D amp Fraser M J 1983 Molecular and Cellular Biology 3 2156 2165 Ayres M D Howard S C Kuzio J Lopez Ferber M amp Possee R D 1994 Virology 202 586 605 Blissard G W amp Wenz J R 1992 J Virol 66 6829 6855 Volkman L E amp Summers M D 1977 J Invertebr Pathol 30 102 103 Monsma S A Oomens A G P amp Blissard G W 1996 J Virol 70 4607 4616 Summers M D amp Smith G E 1978 Virology 83 390 402 Rohrmann G F 1986 J Gen Virol 67 14
26. ensee and the Licensee hereby accepts a limited non exclusive non transferable licence to use the Materials for the Purpose and as otherwise set out in this licence The Licensee warrants to the Licensor that 7 1 it shall only use the Materials for the purpose of Research and 7 2 it shall not alter reverse engineer produce manufacture or amplify the DNA and 7 3 it shall not sell any protein and or virus created pursuant to this Licence to any third party and 7 4 it shall not provide any services to any third party using the Materials and 7 5 if the Licensee desires to the Materials for any purpose other than the Purpose it shall notify the Licensor accordingly and procure a suitable licence prior to any such use The Licensee shall keep the DNA in accordance with the directions contained in the User Guide The Licensor shall raise an invoice to the Licensee for the Fee and the Licensee agrees to pay the same to the Licensor within thirty 30 day of receipt of the invoice unless otherwise agreed in writing The Materials are provided as is and neither the Licensor nor any staff acting on its behalf accepts any liability whatsoever for any of the Materials or in connection with the Licensee s possession handling or use of the Materials The Licensee s remedy pursuant to this Licence shall be limited at the Licensor s option to the replacement of the Materials or a refund of the Fee paid by the Licensee Ownership of the Ma
27. entrifuge to remove any floating cells and decant into a fresh tube If expression levels are expected to be on the low side treat 1 ml of medium with Strataclean resin which concentrates the protein ready for SDS PAGE and or Western blot analysis If the protein is intracellular scrape the cells into the culture medium with a blue Gilson tip pellet the cells ina microcentrifuge tube If liked you can wash the dish with TE buffer to remove the last few cells and add these to the tube with the main bulk of cells Wash the cell pellet with TE buffer and resuspend the cell pellet in SDS PAGE loading buffer and boil samples in the usual way We may later optimise expression by testing expression at multiple time points see 9 3 It is well worth testing expression in both Sf9 Sf21 cells and TniHi5 cells See the OET Cell Culture handbook for details of culturing Tni cells Sometimes there can be a large difference in the expression levels between these two cell lines Whilst Tni cells should not be used for virus amplification due to accumulation of mutations they can be an excellent cell line for protein production and grow well in serum free medium in shake cultures 8 3 Optimisation of expression Sometimes it is necessary to optimise expression levels This is particularly important if you are going to scale up production of protein work here can save litres of medium and hard work later on You can either set up multiple 35 mm dishes
28. erlay Sigma 2 w v solution in d water It is convenient to make up small batches 7 ml of agarose overlay by melting the agarose using a boiling water bath or microwave oven take care Solidified agarose can be stored and re melted prior to use Cool to hand hot before making up final overlay Sterile pipettes and a 12 well plate to make dilutions Beaker with hand hot water as a temporary water bath Plastic sandwich box Incubator at 28 C Phosphate buffered saline PBS Neutral Red stain Sigma 5 mg ml in d water filter sterilize and store at room temperature For use dilute 1 in 20 with PBS solution Do not store diluted stain 22 Page Oxford Expression Technologies flashBAC User Guide 2015 1 Virkon Amtec or similar disinfectant Inverted phase contrast light microscope Lightbox to view plaques Method 1 Seed wells of a 12 well plate with a sub confluent monolayer of healthy Sf21 cells or Sf9 cells if Sf21 are not available See 7 2 1 for more details About 4 x 10 cells well Allow the cells to settle for at least one hour You need 1 x 12 well plate per virus to be titrated Alternatively you can seed 35 mm dishes with cells see 7 2 1 see protocol 7 3 for doing plaque assays in 35mm dishes Sf21 cells in TC100 with 10 serum give the largest easy to spot plaques because these cells have a well defined CPE see Figure 7 Sf9 cells will also yield plaques but they are smaller take longer to dev
29. g the 1 hour incubation period above prepare the co transfection mix of DNA and transfection reagent For each co transfection in either a 35mm dish or well of a 12 well plate you need to mix in a polystyrene tube in the following order 100 ul serum free medium TC100 preferably or serum free growth medium or ES Transfection Medium 100 ng virus DNA from the kit flashBAC or BacPAK6 54l 500 ng transfer plasmid 5 ul acZ transfer vector from flashBAC kit or YOUR transfer vector 1 2 ul baculoFECTIN II Mix total volume 111 2 ul and leave at room temperature for 15 mins Set up a control transfection mix by omitting the DNAs if wished Note This protocol is optimised for using baculoFECTIN II If using a different reagent consult the protocol supplied by the manufacturer 3 If the plated cells were maintained in serum containing medium wash the monolayers twice with TC100 without serum and then add 1 ml of TC100 without serum or ES Transfection Medium to each 35 mm dish or well of a 12 well plate If the cells were maintained in serum free medium 15 Page Oxford Expression Technologies flashBAC User Guide 2015 there is no need to wash at this step simply remove and discard 1ml of medium from the 35mm dishes 4 Allthe 35mm dishes or wells of a 12 well plate should at this stage contain 1 ml of medium without any serum Pipette the 111 2 ul transfection mix from stage 2 into each 35mm dish well of a 12 wel
30. ge at 4 C and so we recommend re titrating virus before use if it has been stored for more than 3 4 months A guide to using flashBAC in 24 well plate systems The following is a guide to making recombinant flashBAC viruses in a 24 well plate format This can be achieved manually or the protocol can be adapted to use in a simple robotic system for liquid handling In this way it is relatively straightforward to make 24 recombinant viruses at one time 24 Page Oxford Expression Technologies flashBAC User Guide 2015 Cells Prepare a master mix of Sf9 cells in serum free medium at a density of 5 x 10 cells ml and dispense 0 4 ml 2 x 10 cells per well Allow cells to settle for one hour Transfection master mix It is convenient to make this in the wells of a 96 well plate Make up a master mix of 220 ul TC100 medium w o serum or other serum free medium and 120 ul flashBAC DNA 5 ul per virus Dispense 14 ul into 24 wells of a 96 well plate Then add 5 ul of the correct transfer plasmid 500 ng DNA and 1 2 ul baculoFECTIN II to each of the 24 wells as appropriate mix by pipetting up and down a few times and allow to stand for 15 min Add transfection mix to cells Simply add the 20 ul transfection mix into each of the wells containing cells in the 24 well plate Seal to prevent evaporation and incubate at 28 C for 5 days Harvest recombinant virus by transferring the culture medium containing budded virus to the wells of a new 24
31. he BEVS is low less than 1 so recombinant virus plaques can often be obscured by parental virus plaques This problem was partially addressed by inserting a copy of the lacZ gene into the virus genome so that recombinant virus plaques would stain blue after the addition of X gal However this did not address the fact that only 1 of plaques went blue and also resulted in contamination of the expressed protein with beta galactosidase 6 3 The BacPAK6 system The efficiency with which recombinant viruses could be recovered was improved by the addition of a unique restriction enzyme site Bsu36l at the polh locus Linearization of the virus genome prior to homologous recombination reduced the infectivity of the parental virus DNA recombinant virus genomes become circular and can replicate This resulted in the recovery of about 30 recombinant virus LacZ was then introduced into the parental virus genome to replace the polh coding sequence resulting in 3 Bsu36l sites at the polh locus Triple digestion of the resulting virus genome with Bsu36l removed a section of virus DNA coding for lacZ and part of the essential gene ORF 1629 resulting in a 9 Page Oxford Expression Technologies flashBAC User Guide 2015 linear virus DNA BacPAK6 that cannot replicate in insect cells Co transfection of insect cells with linearised BacPAK6 DNA and a transfer plasmid with foreign gene under control of polh creates recombinant virus DNA in which ORF162
32. hitinase This enzyme blocks the secretory pathway and its absence helps improve membrane and secreted protein production Backbone virus DNA has gene deletions for chiA and v cath This avoids production of chitinase and cathepsin a viral protease that may otherwise degrade susceptible target proteins See Figure 6 Backbone virus DNA has deletions of chiA v cath and p10 p26 p74 Deletion of p10 results in delayed cell lysis particularly noticeable in TnHi5 cells and thus can extend protein production times It also reduces the metabolic burden on the cell of producing high levels of P10 protein No gene deletions in the virus back bone Useful if the proteins being expressed form complexes inside the cytoplasm or nucleus that need to be purified We find that the relatively early cell lysis associated with PRIME makes it easier to purify these complexes e g VLPs Advantages of the flashBAC system e Simple to use e One step procedure that does not require plaque purification Figure 4 e Amenable to making many viruses simultaneously manually or using a robot in 24 well plates Figure 4 5 e Amenable to high throughput systems e Maximise secreted or membrane targeted proteins Figure 6 e Maximise difficult protein production e Maximise VLP production and release from cells e Back compatible with a large range of transfer plasmids e Now compatible with Gateway cloning system Experimental Overview Co tr
33. ing the agarose overlay it interferes with the gelling process and can produce cracks It may even cause the overlay to fall out when you tip off the stain It may also allow the virus to spread under the overlay and so the plaques appear smeared and diffuse Always add inoculum to the centre of the dish and rock the dish a few times during the incubation period to ensure even coverage of the virus Ensure the cells are also evenly distributed over the dish Do not use a swirling motion at any time as they simply sends cells and virus to the edges of the dish Occasionally multi well plate wells do not have perfectly flat surfaces in our experience the worst culprits for this are 6 well plates and so we always use individual 35 mm dishes Q cannot detect any gene expression A Were the cells used for test expression in good condition see answer above about cells Did you use a virus with a known titre by plaque assay or QPCR there may not have been any virus if you didn t Has the virus been stored for some time before use did you add serum to maintain the titre If not re titrate your virus and try again Only use QPCR on fresh virus If you have a control virus did that work Very occasionally the 27 Page Oxford Expression Technologies flashBAC User Guide 2015 gene is not stable check that the gene is actually in the virus genome by PCR Harvest cellular virus DNA from a 35 mm dish and use for PCR analysis Is the g
34. l plate as appropriate Place in a sandwich box and incubate overnight at 28 C 5 After overnight incubation add one extra ml of growth medium to the 35mm dishes or replace the 1 ml medium in the 12 well plates with 1 ml growth medium Continue the incubation for 4 more days 5 day in total Growth medium may either be serum free medium or TC100 with 10 serum Note Cells in which virus has replicated appear different from mock transfected cells so comparing mock transfected cells with experimental dishes can be a useful indicator that the transfection has worked infected cells appear more grainy with swollen nuclei 6 Harvest the culture medium containing budded recombinant virus into a sterile container and store in the dark at 4 C Note If you prepared a control virus with the lacZ transfer plasmid in the flashBAC kit you can check for transfection success by staining the monolayer of cells left after harvesting the PO virus add 1 ml of growth medium containing 15ul 2 v v X gal to the cell monolayer and leave for a few hours to overnight for the blue colour to develop 7 The next step depends on whether you have used BacPAK6 or flashBAC DNA flashBAC DNA Your 1 2 ml stock of virus is your seed stock PO you now need to amplify this to obtain a 50 ml P1 stock of virus for experimental work and freezing down go to 8 4 BacPAK6 DNA You now need to plaque purify your recombinant virus to obtain your seed stock PO go
35. longer grow properly Always check cells on a regular basis and do not let cultures overgrow If this happens go back to liquid nitrogen stocks are set up a new culture Far more important than passage number of the cell is the number of times the culture has been stressed 26 Page Oxford Expression Technologies flashBAC User Guide 2015 Cells that are not growing well should never be used to make recombinant viruses amplify virus or test for protein production because each of these techniques requires the virus to infect and replicate inside cells and it can only do this is the cells are actively replicating i e in log phase of growth Q My cells are not growing and have enlarged nuclei A See above but they also may be contaminated with baculovirus Start with a fresh cell culture Never use virus and stock cells in the same Class 2 hood Always do cell culture work before virus work Q My co transfection has not worked or become contaminated A See Q on cells above Have you followed the protocol exactly Try a different transfection reagent The plasmid DNA used in the co transfection must be sterile try precipitating with alcohol and re suspending in sterile TE Check you medium is not contaminated The flashBAC and BacPAK6 DNA is quality checked to ensure it is sterile Q My virus has not amplified to high titre A See Q on cells above this is the most likely problem Did you infect cells with low MOI 0 1 pfu cell
36. m the plasma membrane of insect cells containing a membrane fusion protein GP64 Figure 2 A GP64 is acquired when the nucleocapsids bud through the host cell plasma membrane The BV form of the virus is 1000 fold more infectious for cultured insect cells compared to the ODV phenotype and is responsible for cell cell transmission in the early stages of infection It is the BV form of the virus that delivers the foreign gene into the host insect cell for expression Figure 1 A schematic representation of the bi phasic life cycle of baculoviruses resulting in budded virus and occlusion derived virus P PDV polyhedra with occlusion derived virus ECV extracellular virus budded virus In the later stages of the infection cycle large numbers of occlusion bodies OB or polyhedra are formed inside the nuclei of cells Figure 2 B amp C These consist of multiple rod shaped nucleocapsids enclosed within an envelope acquired de novo in the nucleus of cells which then become embedded within a para crystalline matrix of the OB polyhedra The major component of the OB matrix is comprised of a single protein polyhedrin 29 kDa which is produced by the powerful transcriptional activity of the polyhedrin gene polh promoter OBs protect the virus and allow them to survive between hosts in the environment Most baculovirus expression vectors do not produce polyhedra see below for details because the coding sequence for polyhedri
37. n a virus infectivity titration If your P1 virus titre was 1 x 10 pfu ml and you wanted to amplify 500 ml P2 virus you would need to add 1 ml of P1 virus to 500 ml cells at 2 x 10 cells ml MOI 0 1 3 Ensure the cells are shaking at the appropriate rpm for the platform being used If cells are not rotated fast enough they will not be oxygenated and the virus will not replicate Allow the virus to amplify for 3 5 days 4 When the cells appear well infected under the light microscope harvest the culture and remove cells by centrifugation at 3000 rpm for 15 min in a bench top or other slow speed centrifuge 5 Aseptically decant the clarified culture medium into storage containers and store in the dark at 4 C Add serum to 5 for longer term storage We also recommend storing aliquots of 1 2 ml at 80 C 7 Titrate your P1 P2 or P3 virus stock before using the most common reason for poor expression levels is that the virus used to infect the cells had not actually amplified and so the cells were not infected 21 Page Oxford Expression Technologies flashBAC User Guide 2015 You can titre your virus by plaque assay the Gold Standard see 8 5 or by QPCR OET has a convenient QPCR titration kit BaculoQuant or OET provides a fast and cost effective virus titration service contact us at info oetltd com for more details Note Virus can also be amplified in monolayer cultures in T75 or T150 flasks Simply seed the flasks t
38. n has been replaced by that of the foreign gene being expressed under control of the polh promoter This is a useful safety feature because recombinant virus cannot persist in the environment in the absence of polyhedra 7 Page Oxford Expression Technologies flashBAC User Guide 2015 Figure 2 A Rod shaped baculovirus particle B Section through a polyhedron showing occlusion derived virus particles embedded in a matrix of polyhedrin protein C Infected cell in culture showing polyhedral in the enlarged nuclei 6 2 The Baculovirus expression system The baculovirus polyhedrin gene is non essential for virus replication in insect cells grown in culture and this has led to the development of the widely used baculovirus expression vector system first described in 1983 The coding sequence of the polyhedrin gene is replaced by the coding region of the gene to be expressed to produce a recombinant baculovirus in which the powerful polyhedrin gene promoter drives expression of the foreign gene Recombinant baculoviruses produced in this way are polyhedrin or polyhedral negative viruses Figure 3 123 a 46 Figure 3 SDS PAGE analysis of cell extracts from 1 non infected insect cells 2 wild type virus infected cells showing polyhedrin protein at 29kDa and 3 recombinant virus infected cells expressing lacZ beta galactosidase note no p polyhedrin protein is made Cells expressing beta galactosidase are also shown
39. nal 6xHis tags and thrombin cleavage site As pOET1C_6xHis but with reversed MCS Price 132 00 132 00 132 00 132 00 132 00 132 00 Catalogue number 100150 100151 100152 100153 100200 100201 100202 100203 100204 100304 100300 100301 100302 100303 100400 100401 100500 100501 100502 101101 101102 101103 101104 101105 101106 Cat No 300103 300104 300105 300106 Cat No 200101 2001011 2001012 200103 2001031 2001032 5 Page Oxford Expression Technologies flashBAC User Guide 2015 pOET3 10 pg pOET4 10 pg pOETS 10 pg pOET6 BacMAM 10 pg pOET1 Gateway 10g P6 9 gene promoter for late phase expression As pOET3 but with reversed MCS Dual expression with polyhedrin and p10 gene promoters CMV promoter for BacMam mediated transduction of mammalian cells pOET1 transfer plasmid with Gateway technology baculoQUANT products available from OET Ltd Product Details baculoCOMPLETE protein expression kit baculoQUANT all in one kit baculoQUANT All in one virus extraction amp titration kit titrePLUS flashBAC all in one titrePLUS flashBAC all in one titrePLUS flashBAC GOLD all in one titrePLUS flashBAC GOLD all in one titrePLUS flashBAC ULTRA all in one titrePLUS flashBAC ULTRA all in one titrePLUS flashBAC PRIME all in one titrePLUS flashBAC PRIME all in one Cell line products available from OET Ltd Product Sf9 cells Sf21 cells for plaque ass
40. nt virus No selection systems are needed However the virus stock is not homogeneous in the way plaque purified virus is and for very large scale applications or for work that may be taken through regulatory processes we recommend a single round of plaque purification For most purposes however plaque purification is not necessary nor needed This single step procedure greatly facilitates the high throughput production of baculovirus expression vectors via automated systems Figure 5 However it is just as useful for a research lab making one or two viruses in individual dishes It is very useful for the novice The flashBAC system is back compatible with all transfer plasmids based on homologous recombination at the polh locus The OET website has details of most of these and they include single dual triple and quadruple expression plasmids those with purification tags at N and C termini and other promoters including p10 p6 9 ie 1 and CMV for mammalian cells It is not compatible with pFASTBac vectors and the Bac toBac system 10 Page Oxford Expression Technologies flashBAC User Guide 2015 Since the launch of the original flashBAC DNA we have made further modifications to help express difficult to express proteins and the different flashBAC variants are now described flashBAC flashBAC GOLD flashBAC ULTRA flashBAC PRIME Backbone virus DNA has a chiA deletion which prevents production of virus c
41. ntracts Rights of Third Parties Act 1999 shall have no application to this Licence whatsoever and the parties do not intend hereunder to benefit any third party End of Limited Use Licence 2 Kit Contents All reagents and materials provided and referred to in this User Guide are for Research Purposes only flashBAC DNA or BacPAK6 DNA Store at 4 C Control transfer plasmid DNA containing lacZ reporter gene Store at 20 C flashBAC kits only Baculovirus Expression System User Guide Download from www oetltd com Certificate of Analysis Download from www oetltd com nN AeA UNBE MSDS Download from www oetltd com Note Transfection reagent and insect cells are NOT supplied as part of this kit 3 Essential Information and Technical Assistance The information given in this User Guide is accurate to the best of our knowledge It is a practical guide to allow researchers to use the flashBAC and BacPAK6 technology to produce recombinant baculoviruses It is not intended as a comprehensive guide to the baculovirus expression system or insect cell culture Those experienced with the baculovirus expression system may find that they are already familiar with much of the information provided Users are reminded that they may require other licences to use the baculovirus expression system or types of insect cells and it is the responsibility of the user to ascertain ad act on this information For additional help or guidance plea
42. o provide a sub confluent monolayer of cells Remove the medium and add the inoculum to give the correct MOI 0 1 pfu cell use 100 or 200 ul PO virus from 8 2 or 8 3 diluted in medium to 500 ul T75 or 1 ml T150 per flask After 45 mins incubation add 10 15 ml medium T75 or 30 m medium T150 and allow the virus to replicate for 3 5 days until all the cells are well infected The titre of virus amplified in this way is not usually as high as that amplified in shake cultures 7 5 Titration of recombinant virus by plaque assay This is the acknowledged gold standard for determining accurate virus titres The protocol below is one that we have adapted for 12 well plates and is convenient and easy to follow However titres can also be obtained by QPCR and OET sells a convenient BaculoQuant kit for this purpose Alternatively OET offers a service to titrate your viruses by QPCR or plaque assay from as little as 60 per virus contact us on info oetltd com Required Virus to be titrated see 7 4 TC100 growth medium with serum best or serum free growth medium antibiotics Penicillin 10000 units ml and Streptomycin 10000 ug ml in 0 85 saline dilute 1 50 for use may be added to plaque assay medium to help reduce the chance of contamination Culture of Sf21 cells best or Sf9 cells that are in log phase of growth and at least 90 viable 12 well plate or 35mm dishes 6 well plates Low temperature gelling Sea plaque agarose for ov
43. or shall mean Oxford Expression Technologies Ltd Material shall mean the Licensor s product known as flashBAC comprising either or both an agreed quantity of DNA and the relevant User Guide Purpose shall mean the use by the Licensee of the Materials for the production of recombinant proteins and or viruses for Research purposes only Research shall mean the Licensor s systematic search or investigation towards increasing the sum of knowledge in the production of recombinant proteins and or viruses User Guide shall mean the instructions provided with flashBAC to enable the Licensee to produce recombinant proteins and or viruses from the DNA The Licensor and the Licensee have agreed to enter into this Licence on the following terms and conditions The Licensee acknowledges and accepts that by opening and or using the Materials it is agreeing to and accepting these terms and condition If the Licensee does not agree to these terms and conditions it must immediately return all the Materials unused to the Licensor who shall issue a refund for the fee The Licensor has certain knowOhow and has developed a product that can be used to produce recombinant proteins and or viruses and has the right to exploit the product under inter alia patent applications numbered EP1144666 WO0112829 and AU6460800 This Licence shall commence on the date hereof and continue until the DNA has been used or destroyed The Licensor hereby grants to the Lic
44. sation is needed however if you are getting your gene synthesised then it makes sense to optimise for insect cells Many people try and secrete membrane protein domains by removing membrane anchor domains this works sometimes but not always When cloning genes into transfer plasmids note e Check the gene is in the correct orientation with respect to the promoter e Check that the first ATG after the promoter is the start codon you want to initiate translation in the mRNA 13 Page Oxford Expression Technologies flashBAC User Guide 2015 e Check you have a stop codon e Check that any fusion or purification tags are in frame and after any signal peptide sequence that will be cleaved off e Sequence any gene that has been cloned via PCR or gene synthesis Check cloning junctions of genes cloned in using RE digestion and ligation e Ensure final plasmid is sterile as it will be used to transfect insect cells you don t want your cells getting bugged e Mini prep type DNA works OK in transfections Contact us on info oetltd com if you need advice or help with transfer plasmids 7 2 Co transfection of insect cells with BacPAK6 or flashBAC DNA and a transfer plasmid to make a seed stock PO of recombinant baculovirus This method uses cells prepared in individual 35mm cell culture dishes or 12 well plates Protocol 7 6 provides an adaptation of this method for making multiple viruses using 24 well plates This method
45. se refer to the Trouble Shooting Section of this Guide and or the Frequently Asked Questions FAQ section of our website www oetltd com If these resources are unable to help please contact us at info oetltd com and we will be pleased to help where possible All technical assistance provided is given in good faith we cannot take any responsibility whatsoever for any results you obtain by relying on our assistance We make no warranties of any kind with respect to technical assistance or advice we provide 4 Safety Requirements These research products have not been approved for human or animal diagnostic or therapeutic use Procedures described within this User Guide should only be carried out by qualified persons trained in appropriate laboratory safety procedures Always use good laboratory practice when handling this product Warning Safety precautions may be necessary when handling some of the reagents described in this user guide Please refer to the material safety data sheets supplied by the appropriate manufacturer 4 Page Oxford Expression Technologies flashBAC User Guide 2015 5 Ordering Inform flashBAC products available Product flashBAC flashBAC flashBAC flashBAC flashBAC GOLD flashBAC GOLD flashBAC GOLD flashBAC GOLD flashBAC GOLD flashBAC ULTRA flashBAC ULTRA flashBAC ULTRA flashBAC ULTRA flashBAC ULTRA flashBAC selection box 1 flashBAC selection box 2 flashBAC PRIME flashBAC PRIME fl
46. solution in d water It is convenient to make up small batches 15 ml of agarose overlay by melting the agarose using a boiling water bath or microwave oven take care Solidified agarose can be stored and re melted prior to use Larger volumes may also be prepared and melted multiple times Cool to hand hot before making up final overlay Sterile pipettes and bijoux or similar containers for making virus dilutions Sterile Pasteur pipettes Beaker with hand hot water as a temporary water bath Plastic sandwich box Incubator at 28 C Phosphate buffered saline PBS Neutral Red stain Sigma 5 mg ml in d water filter sterilize and store at room temperature For use dilute 1 in 20 with PBS solution Do not store diluted stain X gal 2 v v in DMF to stain for blue plaques 1 Virkon Amtec or similar disinfectant 17 Page Oxford Expression Technologies flashBAC User Guide 2015 Inverted phase contrast light microscope Lightbox to view plaques Method 1 Seed 35mm cell culture dishes with a sub confluent monolayer of healthy Sf21 cells or Sf9 cells if Sf21 are not available See 8 2 1 for more details Allow the cells to settle for at least one hour You need 12 dishes per virus Sf21 cells in TC100 with 10 serum give the largest easy to spot plaques because these cells have a well defined CPE see Figure 7 Sf9 cells will also yield plaques but they are smaller take longer to develop and are not quite so easy to d
47. starting and use aseptic technique throughout 20 Page Oxford Expression Technologies flashBAC User Guide 2015 Required Stock of virus to be amplified e g PO from method 7 2 or 7 3 50 to 200 ml of healthy log phase Sf9 or Sf21 cells at no more than 2 x 10 cells ml Shake flask appropriate to the volume of Sf9 cells to be used you need maximum surface area for oxygen exchange as when cells are infected they have a high O requirement OR Stirred flask e g Techne for Sf21 cells in medium containing serum Growth medium serum free or TC100 with 10 serum Incubator at 28 C with shaking platform or Stirred culture platform Phase contrast light microscope Disinfectant for discard Sterile pipettes Method 1 Prepare 50ml to 200 ml log phase Sf9 cells or Sf21 cells in a shake or stirred culture as appropriate to the medium being used Generally Sf9 cells in serum free medium in a shake culture should not be more than 2 x 10 per ml and Sf21 cells in serum containing culture should not be more than 1 x 10 cells ml 2 To amplify virus simply add the appropriate volume of inoculum to give a low MOI of 0 1 pfu cell When amplifying the seed stock PO of flashBAC virus from 8 2 or BacPAK6 from 7 3 we recommend adding no more than 0 5 ml virus into 100 ml culture we do not normally titre the seed stock virus before P1 amplification If we are amplifying P1 to P2 or P2 to P3 we always use a defined amount of inoculum based o
48. terials shall pass to the Licensee upon dispatch of the Materials by the Licensor to the Licensee The Licensee shall indemnify the Licensor for any loss suffered by the Licensor as a result of the Licensee s breach of this licence and or third party s intellectual property rights This Licence is personal to the parties and shall not be assigned or otherwise transferred in whole or in part by either party This Licence constitutes the entire agreement and understanding between the parties in respect of the Materials and supersedes all previous agreements understandings and undertakings in this respect and all obligations implied by the law to the extent that they conflict with the express provisions of this Licence 3 Page Oxford Expression Technologies flashBAC User Guide 2015 16 The invalidity illegality or unenforceability of a provision of this Licence shall not affect or impair the continuation in force of the remainder of this Licence 17 The Licensor reserves the right to revoke this permission and may require the Licensee to return or destroy any remaining DNA and or the User Guide 18 Clauses 1 3 7 9 10 13 16 18 20 shall survive any termination or expiry of this Licence 19 The interpretation construction and effect of this Licence shall be governed and construed in all respects in accordance with the laws of England and the parties hereby submit to the non exclusive jurisdiction of the English courts 20 The Co
49. the system over the last few years Our focus is on the improvements made with the system we call flashBAC which was developed to make it easier and quicker to make recombinant viruses and to help achieve better expression with difficult to express proteins Generally the baculovirus genome is considered too large to insert genes directly although one commercial product BaculoDirect achieves this Instead foreign genes are cloned into a transfer plasmid which contains sequences that flank the polyhedrin gene in the virus genome The virus genome and transfer plasmid are simultaneously introduced into insect cells co transfection and homologous recombination between the flanking sequences of the polh in the plasmid and genome results in exchange of DNA resulting in a recombinant baculovirus Figure 3 The virus genome then replicates and produces recombinant virus which can be harvested as budded virus in the culture medium In most available BEVS using the homologous recombination method this results in a mixture of recombinant virus and recirculation of the parental virus DNA to produce non recombinant virus These are separated by plaque purification to produce a stock of pure recombinant virus Plaque purification is time consuming and technically demanding to the non virologist Many developments have attempted to improve the method by which recombinant and parental virus may be separated The frequency of recombinant efficacy in t
50. to 2 w v by boiling water bath or microwave oven take appropriate safety precautions You need 1 ml per dish of cells Cool the overlay to hand hot about 50 55 C and add an equal volume of pre warmed growth medium 28 C Keep warm to prevent setting we use a temporary clean water bath comprising a beaker of hot tap water You need 2 ml final overlay per dish If the agarose in water sets it is easy to melt again by boiling If the agarose overlay with growth medium sets you cannot re melt You have to start again We often prepare several small batches of agarose in water and let them set and then melt each aliquot as we need it 15 ml is convenient 6 At the end of the incubation period 4 remove the inoculum using a pipette and discard into Virkon or other disinfectant Working quickly add 2 ml warm overlay to each dish allowing the agarose to flow down the side of the dish and spread slowly over the monolayer of cells Do NOT pipette into the centre of the dish 18 Page Oxford Expression Technologies flashBAC User Guide 2015 10 11 12 13 Process one set of dishes per virus sample at a time If working ina hood keep the agarose overlay in a beaker or sandwich box filled with warm water to delay solidification If the agarose sets prematurely you can leave the dishes with virus inoculums for longer than 60 min without adverse effects If you have removed the virus and then find that your overlay medium has set
51. to 8 3 Note You can also plaque purify virus produced using flashBAC DNA if required go to 8 3 16 Page Oxford Expression Technologies flashBAC User Guide 2015 7 3 Plaque purification of recombinant BacPAK6 virus The budded virus harvested after the co transfection with BacPAK6 virus DNA will contain a mixture of parental virus about 10 blue and recombinant virus about 90 clear colourless These need to be separated by performing a plaque assay and picking individual plaques to amplify pure virus stocks As long as well isolated plaques are picked only one round of plaque purification is needed This is a multi step method that enables you to isolate plaques and then amplify plaque picked virus to produce a PO seed stock of virus Read through the whole method before starting to ensure you are aware of time scales and reagents equipment needed at each stage The OET Cell Culture Manual has details of insect cell culture Required Virus harvested from a co transfection see 7 2 TC100 growth medium with serum best or serum free growth medium antibiotics Penicillin 10000 units ml and Streptomycin 10000 ug ml in 0 85 saline dilute 1 50 for use may be added to plaque assay medium to help reduce the chance of contamination Culture of Sf21 cells preferred or Sf9 cells that are in log phase of growth and at least 90 viable 35 mm dishes and T25 flasks Low temperature gelling Sea plaque agarose for overlay Sigma 2 w v
52. ul construct plans are needed to insert genes according to MCS and restriction sites available These contain a mammalian promoter in place of polyhedrin so that the final recombinant virus can be used to effect gene expression in mammalian cells e g pOET6 CMV promoter Use baculovirus gene promoters that are expressed earlier than polyhedrin and P10 in the late phase of gene expression Useful for secreted or membrane targeted protein where polyhedrin P10 has not worked or for proteins that need processing before the virus shuts off host cell protein production E g pOET3 and 4 use the p6 9 gene promoter Several transfer plasmids will give the option for N or C terminal tags such as His Strep HA to aid protein purification These may also have a cleavage site to release the final product from the tag Some transfer plasmids allow the gene to be expressed as a fusion product with a fluorescent protein for example to allow visualisation by microscopy Generally the natural signal peptide of a protein will work in insect cells but if you want to add a signal peptide or use an insect virus one then the signal peptides of either AcMNPV GP64 or chitinase work very well Adding a signal peptide to a protein that is not normally secreted may not work Translation will start at the first ATG after the promoter so check constructs carefully to ensure there is no inadvertent additional ATG There is no general data to show that codon optimi
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