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1. 3 If you want the target protein to be secreted or directed to the membrane the inserted gene should have the appropriate signal peptide and hydrophobic anchorage encoding seguences Many mammalian sig nals are recognized in insect cells 4 The target gene should not contain introns use cDNA 5 The 5 untranslated leader sequence should be as short as possible remove leader regions with a high GC content or stable secondary structures if possible 6 Transcription of the inserted gene is terminated by the polyhedrin polyadenylation signal in the transfer vector B Inserting the Target Gene into the Transfer Vector 1 Clone the insert into the appropriate site of pBacP AK8 or pBacPAK9 or other suitable vector 2 Screen for transfer vectors having the insert in the correct orientation by PCR amplification using the 2801 and Bac2 Primers or by perform ing restriction digests of mini prep DNA 3 Optional Confirm the integrity of the junctions by sequencing with the 2801 and Bac2 Primers 4 Prepare plasmid DNA by CsCl isopycnic density gradient centrifuga tion or by alkaline lysis miniprep followed by purification with a CHROMA SPIN TE 400 Column You may also use a NucleoBond Plasmid Purification Kit K3000 1 2 3 CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 18 Version PR95847 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Viral Expression V
2. 30 ml Spinner shake flasks 2 0 105 ml 50 500 ml CLONTECH Laboratories Inc www clontech com Protocol 1 12 Version PR95847 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued E Suspension Cultures of Sf21 Cells Suspension cultures using either spinner or shake flasks are easy to maintain and reproducibly give cells of a high viability gt 95 that are good for experimental work Suspension cultures are particularly useful when large numbers of cells are needed Growing cells in spinners requires a low speed magnetic stir platform which can be placed inside a 27 C incubator Note that some stir platforms generate too much heat to be used inside an incubator Flat bottomed pyrex flasks 100 1000 ml containing a magnetic stir bar and covered with a foil cap can be used as spinner flasks Alternatively suspension cultures can be grown in shake flasks using an orbital shaker normally used for bacterial cultures 1 Add an appropriate volume of prewarmed BacPAK Complete Medium to a sterile spinner or shake flask Inoculate with cells to give a starting density of 4 x 10 cells ml Note Insect cells have a high oxygen demand therefore suspension cultures must have a high surface area to volume ratio or cell growth will be inhibited The culture volume should be no more than two fifths of the total volume of the flask e g 40 ml of medium in a 100 ml flask or
3. Baculovirus Expression System User Manual IX Virus Propagation and Evaluation continued 6 Analysis of virus infected cell DNA a Resuspend the cell pellet in 250 ul of TE buffer b Add 250 ul of lysis buffer 12 5 ul of 10 mg ml proteinase K and 2 ul of 10 mg ml RNase Incubate at 37 C for 30 min c Add 500 ul of phenol chloroform 50 50 Mix by inversion for 5 min Spin in a microcentrifuge for 2 min to separate the phases Move the aqueous layer to a fresh tube repeat the extraction twice d Transfer the aqueous layer to a fresh tube add 50 ul of 3 M sodium acetate and 1 mlof ethanol Chill at 20 C for 10 min Pelletthe DNA in a microcentrifuge at room temperature for 5 min Add 0 5 ml of 75 ethanol vortex briefly and then repellet the DNA Repeat the ethanol wash Dry the pellet at room temperature for 30 min e Add 50 ul of TE buffer to pellet and soak at 4 C overnight Gently resuspend DNA using a pipette tip You may digest aliquots ofthis DNA with a restriction enzyme and analyze by Southern blotting C Processing and Storage of the Passage One Virus Stock 1 After confirming that the plaque comrpises recombinant virus containing the target gene transfer 1 ml of recombinant virus Passage One stock to 709C for long term storage Cryogenic agents are not required 2 Determine the titer ofthe Passage One stock of recombinant virus Section IX E You may use the BacPAK Baculovirus Rapid Titer Kit K1599 1 or
4. Non Exclusive Rights to use Baculovirus Expression vector system technology for research purposes Background The Texas Agricultural Experiment Station TAES claims rights to technology developed by Dr Max D Summers of the Department of Entomology relating to a recombinant baculovirus expression vector system BEVS and the use of such vectors in insect cell culture media for expression of cloned genetic material TAES is making the system and its components available for noncommercial research purposes This baculovirus expression vector system and related subject matter are claimed in two United States Patents Numbers 4 745 051 and 4 879 236 Commercial rights to BEVS or products thereof are subject to a non exclusive license terms of which will be made available upon written request Information and materials received from TAES relating to BEVS must be taken with the understanding that it is subject to a restrictive license for research purposes only Terms and Conditions of Agreement 1 All information and material received under this Agreement shall be used for research purposes only 2 Access to and distribution of the vectors and information must be limited to the Recipient and to those personnel who report to the Recipient hereinafter referred to as Recipient 3 Recipient agrees to supply TAES preprints of any publications resulting from the use of the BEVS material promptly upon receipt by Recipient of notice of accepta
5. 123 Kitts P A amp Possee R D 1993 A method for producing recombinant baculovirus expression vectors at high frequency BioTechniques 14 5 810 817 Luckow V A 1991 Cloning and expression of heterologous genes in insect cells with baculovirus vectors in Recombinant DNA Technology amp Applications eds Prokop A Bajpai R K amp Ho C S McGraw Hill Inc NY pp 97 152 Luckow V A amp Summers M D 1988 Trends in the development of baculovirus expression vectors Bio Technology 6 47 55 Malitschek B amp Schartl M 1991 Rapid identification of recombinant baculoviruses using PCR BioTechniques 11 177 178 Miller L K 1988 Baculoviruses as gene expression vectors Ann Rev Microbiol 42 177 199 OReilly D R Miller L K 8 Luckow V A 1992 Baculovirus Expression Vectors A Laboratory Manual W H Freeman amp Co NY Possee R D 1986 Cell surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector Virus Res 5 43 59 Possee R D Sun T P Howard S C Ayres M D Hill Perkins M amp Gearing K L 1991 Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9 4 kbp EcoR l I and R polyhedrin gene region Virol 185 229 241 Richardson C D 1995 Baculovirus Expression Protocols Methods in Molecular Biology Volume 39 Humana Press NJ Sisk W P Bradley J D Seivert L L Vargas R A amp
6. Asp718l Amp pBacPAK9 Fholyhediin eh g 5 5 kb ark signal RS BstB p Xho l Hind Ill Stul 1764 Pstl Sse8387 I Eagl Dra lll 3075 Not Pac 1 1325 1250 bp AcMNPV Scal 3990 1433 bp M13 ori AcNPV Figure 5 Map of pBacPAK9 Transfer Vector 1151 A Bac1 Primer TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA EcoR V 201 e polyhedrin promoter AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA 1 1251 CGGATCCCGG GAATTCGAGC TCGGTACCAG ATCTTCTAGA TICGAACTCG BamH I EcoRI Echl36llAsp 181 Bglll Xbal BstBl Xhol Xmal Sac Kpn I Smal 1301 stops s ITN AGGCCTGCAG GGCGGCCGCT TAATTAATTG ATCCGGGTTA TTAGTACATT Stul Eagl Pacl se83871 Not an PstI TATTAAGCGC TAGATTCTGT GCGTTGTTGA TTTACAGACA ATTGTTGTAC GCG ATCTAAGACA CGCAAC SnaB lt lt m Bac2 Primer 1401 GTATTTTAAT AATTCATTAA ATTTATAATC Figure 6 Sequences in and around the pBacPAK9 multiple cloning sites Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 31 BacPAK Baculovirus Expression System User Manual Appendix A Vector Maps and MCS Seguences continued BamH 5508387 Pst Stul Poolyhedrin mp pBacPAK8 GUS 7 4 kb EcoR Xma Smal Eagl Notl Pacl poly At signal 1433 bp AcMNPV M13 ori Figure 7 Map of pBacPAK8 GUS Transfer Vector Notice to Purchaser This product is optimiz
7. Horlick R A 1992 An improved method for rapid screening of baculovirus recombinant plaques by PCR amplification BioTechniques 13 2 186 Vaughn J L Goodwin R H Tompkins G J amp McCawley P 1977 The establishment of two cell lines from the insect Spodoptera frugiperda Lepidoptera Noctuidae n Vitro 13 213 217 Vlak J M amp Keus R J A 1990 Baculovirus expression vector system for production of viral vaccines in Viral Vaccines Wiley Liss Inc NY pp 91 128 Webb A C Bradley M K Phelan S A Wu J Q amp Gehrke L 1991 Use of the polymerase chain reaction for screening and evaluation of recombinant baculovirus clones BioTechniques 11 512 519 CLONTECH Laboratories Inc www clontech com Protocol lt 1 Version PR95847 BacPAK Baculovirus Expression System User Manual Catalog 6144 1 6145 1 6146 1 6142 1 K1601 A K1601 B K1601 C K1601 E 8090 1 8091 1 V2188 1 K1599 1 K3000 1 2 3 CLONTECH Laboratories Inc 29 www clontech com XIII Related Products Product BacPAK6 DNA Bsud6 digest pBacPAK8 Transfer Vector pBacPAK9 Transfer Vector pAcUW31 Transfer Vector 0801 Primer Bac2 Primer BacPAK6 Virus Stock IPLB Sf21 Cells BacPAK Complete Medium BacPAK Grace s Basic Medium Baculovirus Expression Protocols BacPAK Baculovirus Rapid Titer Kit NucleoBond Plasmid Purification Kit Protocol PT1260 1 Version PR
8. However we recommend picking up to 10 well isolated plaques Test three to four of these putative recombinant viruses for the target gene and store the remainder at 4 C Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 23 BacPAK Baculovirus Expression System User Manual IX Virus Propagation and Evaluation A Preparation of Passage One Virus Stock Use plague picks to generate virus infected cell proteins or DNA This step also amplifies the virus in the plague pick 1 Seed 35 mm dishes one for each plague pick with 5 105 Sf21 cells in 1 5 2 5 ml of BacPAK Complete Medium Incubate at 27 C for 1 6 hr 2 Remove medium from cells Gently add 100 ul of a plaque pick near the center of the dish As controls plate 100 ul of medium and 100 wl of a 10 dilution of the BacPAK6 parental or AcMNPV C6 wild type virus stocks 3 Incubate at room temperature for 1 hr Add 2 ml of BacPAK Complete Medium to each dish 5 Incubate at 28 C for 3 4 days until the cells look well infected Infected cells may be grainy sausage shaped or have rough borders 6 Transfer medium to a sterile centrifuge tube Do not discard the dish you will use the cells later 7 Centrifuge medium at 1000 x g for 5 min to remove cells and debris 8 The supernatant is the Passage One virus stock Transfer it to a fresh sterile tube Store at 4 C See Section IX C 1 for further processing of the Passage
9. Method Plague assays are designed to produce distinct viral plagues in a monolayer of host cells under conditions where each plaque is the result of a cell being infected by a single virus Plague assays can thus be used to isolate an individual recombinant virus from the pool of viruses generated by a cotransfection Section VIII C Plague assays can also be used to determine the titer of a virus stock however titers can be obtained more guickly and easily using CLONTECH s BacPAK Baculovirus Rapid Titer Kit K1599 1 If plaque assays are to be used either to produce a pure recombinant virus clone or to titer virus stocks it is advisable to develop good plaque assay technique by practicing using the virus stocks provided A Practice Plaque Assay BacPAK6 is a convenient virus for practice because it expresses B galactosidase Kitts amp Possee 1993 and produces plaques that can be stained blue with X gal 1 Remove an aliquot of exponentially growing Sf21 cells that have a viability of gt 95 and dilute with prewarmed BacPAK Complete Me dium to make 18 ml of cell suspension at 7 x 10 cells ml 2 Add 1 5 ml of the cell suspension to a 35 mm tissue culture dish and rock to distribute evenly Repeat for 9 more dishes Each dish will receive approximately 1 x 105 cells Incubate the cultures at 27 C for 1 4 hr Notes The correct cell density is critical to assay success To minimize problems with medium evaporation from t
10. amp V E Plasmid DNA Viral DNA Section V F z Practice plaque assay Section VLA Cotransfect Sf21 cells with plasmid DNA and BacPAK6 viral DNA Section VIII A U Plague assay of progeny viruses Section VIII C amp VI A Pick several putative recombinant virus plaques amp Confirm presence and or expression of target gene Sections IX A amp B Yv Amplify recombinant virus You may use theBacPAK rs Sections IX C 8 D Baculovirus Rapid Titer Kit K1599 1 at steps IX C amp IX E a v Titer amplified virus stock Section IX E v Characterize gene expression Section X Scale up protein production Section XI Figure 2 Schematic diagram outlining BacPAK Baculovirus Expression Procedures Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 9 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines A General Considerations The IPLB Sf21 cell line abbreviated Sf21 originally derived from the fall army worm Spodoptera frugiperda Vaughn et al 1977 is used to propagate AcMNPV based expression vectors These cells grow reasonably well from room temperature 22 C to 30 C and do not require COs At their optimum growth temperature 27 C their doubling time is 20 24 hr Although you can culture Sf21 cells on the bench maintaining them in an incubator at 27 C is preferable for consist virus infect
11. should have viabilities of 80 90 monolayers harvested from glass flasks should have viabilities of gt 90 and suspension cultures should have viabilities of gt 95 14 Remove all but 2 ml of the 10 ml cell suspension and store it in a sterile container Add 30 ml of prewarmed BacPAK Complete Medium to the remaining 2 ml of culture Swirl to mix and incubate at 27 C For information on incubation times see note below 15 Add 2 ml of the reserved cell suspension to a second 150 cm flask containing 30 ml of prewarmed BacPAK Complete Medium Swirl to mix and incubate at 27 C The cells from this flask will be frozen Section V F For information on incubation times see note below 16 Use a portion of the remaining reserved cell suspension to seed a 50 ml suspension culture Section V E Maintain monolayer stocks of cells by repeating Steps 8 14 You must passage monolayers split 1 8 and grown at 27 C every 3 4 days you must passage monolayers split 1 10 and grown at room temperature once a week Depending upon your needs you may split near confluent monolay ers at any ratio between 1 2 and 1 20 As needed seed additional monolayer flasks and suspension cultures to provide cells for experiments For additional information on seeding densities see Table I TABLE GUIDELINES FOR SEEDING DENSITIES Size of vessel Number of cells Volume of media 25 cm flask 1 0 x 108 5 ml 75 cm flask 3 0 x 108 10 ml 150 cm flask 6 0 x 10
12. 100 ml in a 250 ml flask 2 Incubate cells at 27 C and stir or shake at 50 100 rpm use the minimum speed that will Keep the cells in suspension 3 Monitor the cell density daily until the culture reaches 2 3 105 cells ml 4 days Add 0 3 ml of cell suspension to 0 3 ml of 0 08 w v trypan blue in PBS Count the cells using a hemocytometer viable cells exclude trypan blue whereas dead cells take up the blue stain 4 Use the cells to seed a fresh spinner shake flask at a density of 1 2 x 10 cells ml Alternatively remove the excess cells and add fresh media to bring the density down to 1 2 10 cells ml 5 Return to stirrer shaker at 27 C and monitor cell density daily as above Notes Maintain one or more suspension cultures to provide cells for experimental work Cells should only be used for virus infections and plague assays if they are in the exponential phase of growth usually 0 7 1 x 108 cells ml Periodically every 6 weeks replace the working suspension cultures with fresh ones started from monolayer cells Take good care of your cells the guality of virus plagues and the level of protein production are very dependent on the health of the host cells Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 13 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued F Storing Insect Cells in Liquid Nitrogen Freezing a
13. 95847 BacPAK Baculovirus Expression System User Manual Appendix A Vector Maps and MCS Seguences AlwN I BstX I MCS 4950 314 BamH 1 1253 Sse8387 Pstl Stul Xhol BstB Frolyhedrin ana pBacPAK8 Bgl ll poly A uo Kpal signal S Ech36 Il 1433 bp Hinam Sacl AcMNPV 1764 EcoR Xma Smal Eagl Dra lll Noti 3075 Pac 1325 AcMNPV M13 ori Figure 3 Map of pBacPAK8 Transfer Vector 1151 i 8801 Primer TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA EcoRV 1201 polyhedrin promoter a AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA 1 1251 CGGATCCCTG CAGGCCTCGA GTICGAATCT AGAAGATCTG GTACCGAGCT BamH Stul BstBl Xbal Bglll Asp7181 Ech36 II Sse8387 Xho Kpni Saci Pstl 1301 stops ATS CGAATTCCCG GGCGGCCGCT TAATTAATTG ATCCGGGTTA TTAGTACATT EcoR Eagl Pacl Xmal Notl Smal 1351 TATTAAGCGC TAGATTCTGT GCGTTGTTGA TTTACAGACA ATTGTTGTAC GCG ATCTAAGACA CGCAACA SnaB I lt lt m Bac2 Primer 1401 GTATTTTAAT AATTCATTAA ATTTATAATC Figure 4 Seguences in and around the pBacPAK8 multiple cloning sites CLONTECH Laboratories Inc www clontech com Protocol lt 1 Version PR95847 BacPAK Baculovirus Expression System User Manual Appendix A Vector Maps and MCS Seguences continued AlwN I BstX I MCS 4950 314 BamH 1 1253 Mlul Xmal 546 Smal EcoR Ech36 ll Sac
14. CLONTECH Innovative Tools to Accelerate Discovery BacPAK Baculovirus Expression System User Manual PT1260 1 PR95847 Published 14 May 1999 Catalog K1601 1 See List of Components for storage conditions FOR RESEARCH USE ONLY o Nu na 10 10 11 11 13 14 15 15 17 17 18 18 18 19 19 21 22 24 24 24 25 25 26 27 27 BacPAK Baculovirus Expression System User Manual Table of Contents Introduction List of Components Additional Materials Reguired Experimental Outline Insect Cell Culture Guidelines A General Considerations Culture Media Establishing the Sf21 Cell Line Subculturing Sf21 Monolayers Suspension Cultures of Sf21 Cells m O Storing Insect Cells in Liquid Nitrogen Plaque Assay Method A Practice Plaque Assay B Calculation of Virus Titer C Troubleshooting Plaque Assays Construction of a Recombinant Transfer Vector A Tailoring the Insert B Inserting the Target Gene into the Transfer Vector Construction of a Recombinant Viral Expression Vector A Generating a Recombinant Virus B Troubleshooting Guide C Isolating Recombinant Viruses Virus Propagation and Evaluation A Preparation of Passage One Virus Stock B Evaluation of Recombinant Viruses C Processing and Storage of the Passage One Virus Stock D Amplification of Recombinant Viruses Preparation of Passage Two Virus Stock E Titration of Amplified Virus Stocks Characterizing Recombinant Gene E
15. One virus stock B Evaluation of Recombinant Viruses Many screening methods can confirm that plaques picked from the cotransfection contain recombinant baculovirus The probes available will dictate the method you choose The preferred methods detect synthesis of the target protein e g Western blotting ELISA or a biochemical assay for the expressed protein If an antibody is not available Southern blotting with a nucleic acid probe or PCR with the Bac1 and 2802 Primers O Reilly et al 1992 Webb et al 1991 Malitschek amp Schartl 1991 Sisk et al 1992 can confirm that the target gene is in the viral genome Detailed screening protocols will not be presented here as they follow standard methods 1 After removing medium from the virus infected cell cultures Step IV A 6 add 1 ml of PBS to each dish and scrape cells into the buffer 2 Pellet the cells in a microcentrifuge at 1 000 rpm for 1 min 3 Remove supernatant and gently resuspend cells in 0 5 ml of PBS 4 Repellet and discard supernatant Analyze cell proteins or DNA as follows 5 Analysis of virus infected cell proteins a Resuspend the cell pellet in 50 100 ul of PBS b Add an appropriate volume of SDS PAGE dissociation mix and boil for 5 min You may run an aliguot of the denatured proteins on a standard polyacrylamide gel and analyze by Western blotting CLONTECH Laboratories Inc www clontech com Protocol lt 1 24 Version PR95847 BacPAK
16. PV C6 Wildtype Virus Stock For long term storage store the following reagents at 209C For storage less than 6 months store at 4 C e 15 ug pBacPAK8 Transfer Vector 500 ng ul e 15 ug pBacPAK9 Transfer Vector 500 ng tu e 2 5 ug pBacPAK8 GUS Vector 100 ng l e 20 ul Bact Primer 20 uM e 20 ul Bac2 Primer 20 uM Note The following kit components are also available separately e BacPAK6 Virus Stock K1601 C IPLB SF21 Cells K1601 E Baci Primer K1601 A Bac2 Primer K1601 B BacPAK6 DNA Bsu digest 6144 1 pBacPAK8 Transfer Vector 6145 1 pBacPAK9 Transfer Vector 61 46 1 CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 6 Version PR95847 BacPAK Baculovirus Expression System User Manual lll Additional Materials Reguired The following materials are reguired but not supplied BacPAK Complete Medium 8090 1 You may also use TNM FH insect cell medium Grace s medium supplemented with yeastolate and lactalbumin hydrolysate with fetal bovine serum cell culture grade ask vendor for a lot tested with insect cells and antibiotics BacPAK Grace s Basic Medium 8091 1 CHROMA SPIN TE 400 Columns K1323 1 Dimethylsulfoxide DMSO cell culture grade Neutral red stain 0 33 Trypan blue dye 0 4 X GAL 25 mg ml 5 bromo 4 chloro 3 indolyl B D galactopyranoside in dimethylformamide DMF Store away from light at 209C X GLUC 25 mg ml 5 bromo 4
17. a plaque assay The titer should be in the range 1 5 x 107 pfu ml D Amplifying Recombinant Viruses Preparing Passage Two virus Stock 1 Seeda50 ml suspension culture with x 10 cells ml and incubate at 27 C until the cell density reaches 4 5 x 10 cells ml 2 days 2 Calculate the volume of the Passage One virus stock that will contain 0 1 pfu for every cell in the suspension culture Multiplicity of Infection M O I 0 1 and add this volume to the suspension culture 3 Incubate at 27 C for 4 6 days until the cells are well infected Centrifuge infected cells at 1 000 x g for 5 min to remove cells and debris 5 Transfer the supernatant to a fresh sterile tube This is the Passage Two virus stock Freeze 5 x 5 ml aliquots at 70 C for long term storage Store the remainder at 4 C and use as the current working stock 6 Determine the titer of the Passage Two stock Section IX E The titer should be gt 5 x 107 pfu ml 7 When the current working stock is depleted thaw an aliquot of Passage Two stock and generate a new working stock by infecting a 50 200 ml suspension culture Steps 1 6 above Note Do not passage the virus repeatedly baculoviruses can accumulate mutations T gt Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 25 BacPAK Baculovirus Expression System User Manual IX Virus Propagation and Evaluation continued E Titration of Amplified Virus Stocks You
18. al with 70 ethanol to decontami nate the outside 5 In a laminar flow hood transfer the cell suspension to the pre warmed flask Incubate at 27 C for 1 3 hr to allow cells to attach Do not incubate for more than 12 hr 6 When a significant fraction of the cells have attached gently remove the medium and replace with 5 ml of fresh prewarmed 27 C medium 7 Incubate at 27 C until a nearly confluent monolayer forms 7 days We recommend checking the flasks for confluency every other day D Subculturing Sf21 Monolayers 1 Examine cell monolayers under an inverted microscope to check that the cells are healthy and ready for passaging The monolayer should be 80 90 confluent Notes e Sf21 cells are not susceptible to contact inhibition If monolayers become overconfluent the cells will start to float and divide in the media Healthy cells should be rounded have distinct cell boundaries and should not appear granular Signs of unhealthy cells are a large number of floating cells before confluency sausage shaped cells stopped in mid cell division and cells with rough boundaries Contaminated cultures will become cloudy within 24 48 hr 2 Remove the old medium and any floating cells from the flask If the cells are mainly detached omit this step and go to Step 4 3 Add 5 ml of prewarmed BacPAK Complete Medium 4 Gently dislodge the cells using a sterile scraper Note Many commercial scrapers are harsh and using th
19. at 27 C for 2 3 hr 15 Aspirate off the stain invert the dishes and leave them in the dark at room temperature overnight to allow the plaques to clear and the blue color to fully develop Notes Neutral red is taken up by healthy cells but not by dead cells Therefore on the dishes stained with neutral red only plaques will be clear circles about 0 5 3 mm in diameter against a red or pink background On the dishes stained with X gal and neutral red plaques will be blue against a red background You should see blue foci in these dishes even if the plaques are small Practice the plaque assay until you can see plaques with neutral red stain alone Neutral red is light sensitive and will become grainy upon exposure to light CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 16 Version PR95847 BacPAK Baculovirus Expression System User Manual VI Plague Assay Method continued B Calculation of Virus Titer 1 Count the plaques on each dish that has a reasonable number of plaques i e 10 30 per dish from this count calculate the average number of plaques per dish 2 Since 0 1 ml of inoculum was applied to each dish the titer of the virus stock pfu ml is average plaques per dish x 10 x dilution factor 3 Example calculation 25 plaques x 10 x 105 C Troubleshooting Plaque Assays To get good plaque formation it is important to use cells which are in the exponential phase of growth a
20. carries the missing ORF1629 sequence andif the large fragment recombines with it the resulting circular DNA will contain all the genes necessary for viral replication This double recombina tion event restores the essential gene and transfers the target gene from the transfer vector to the viral genome Cotransfections using 85036 0 BacPAK6 viral DNA produce recombinant viruses at frequencies approaching 100 This User Manual contains directions for establishing insect cell cultures as well as for isolating a recombinant baculovirus expression vector using the BacPAK system More extensive protocols for using baculovirus expression systems are in the baculovirus laboratory manuals O Reilly et al 1992 King amp Possee 1992 Richardson 1995 CLONTECH V2188 1 Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 5 BacPAK Baculovirus Expression System User Manual ll List of Components Store the following item at 180 C liquid nitrogen immediately upon receipt 2 X 108 IPLB Sf21 Insect Host Cells in TNM FH 1096 FBS 10 DMSO Store the following items at 49C do not freeze The following components are sufficient for five transfections e 25 ul BacPAK6 Viral DNA Bsu36 I digest e 25 ul Bacfectin For long term storage of 6 months or longer store the following reagents at 70 C For storage less than 6 months store at 4 C e 2 ml BacPAK6 Virus Stock e 2 ml AcMN
21. chloro 3 indolyl B D glucuronic acid CLONTECH 8080 1 in DMSO Store away from light at 209C RNase A 10 mg ml Store at 209C Proteinase K 10 mg ml made fresh 4031 1 Store at 0 SeaPlaque Agarose FMC BioProducts 50101 Sterile H O 3 M NaOAc pH 5 2 Lysis buffer 50mM Tris HCI pH 8 0 10mM EDTA 5 _ B mercaptoethanol 0 4 Sodium dodecylsulfate Phosphate buffered saline PBS 140mM NaCl 27mM_ KCI 8mM Na HPO 1 5 mM _ KH PO pH 7 3 TE buffer 10 mM Tris HCl pH 8 0 1mM EDTA Phenol chloroform 50 50 equilibrated with 100 mM Tris HCl pH 8 0 Ethanol 100 and 70 Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 7 BacPAK Baculovirus Expression System User Manual IV Experimental Outline Please refer to Figure 2 on the following page e Obtain insect cell media and establish Sf21 cell line This step will take 3 4 weeks Section V B C Maintain working stocks of Sf21 cells Sections V D E e When the stock of cells has been passaged twice freeze aliquots for long term storage in liquid nitrogen Aliquots of frozen cells provide a back up in case the working stock dies or becomes contaminated Frozen cells are also a source of fresh cells for replacing working stocks as they become old Section V F e Practice assaying viral plaques using the BacPAK6 virus stock provided in the kit Section VI A Isolating pure recombinant virus requires good vira
22. chnology which recipient can show are in the public domain or which he she had previously received or developed in good faith through channels independent of the Texas Agricultural Experiment Station 1 Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 BacPAK Baculovirus Expression System User Manual Notes CLONTECH Laboratories Inc www clontech com Protocol lt 1 34 Version PR95847 BacPAK Baculovirus Expression System User Manual Notes Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 35
23. d the corresponding sequences in the viral DNA transfers the target gene to the viral genome The BacPAK Baculovirus Expression System uses BacPAK6 a specially engineered virus that facilitates construction and selection of recombinant expression vectors BacPAK6 has an essential gene adjacent to the polyhedrin locus that provides selection for recombinant viruses Kitts amp Possee 1993 Figure 1 Sites for 25096 which does not cut wild type AcMNPV DNA were introduced into the genes flanking the polyhedrin expression locus of BacPAK6 Digesting BacPAK6 with Bsu36 releases two fragments The first carries part of a downstream gene ORF 1629 that is essential for viral replication Possee CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 4 Version PR95847 BacPAK Baculovirus Expression System User Manual l Introduction continued f ori amp Transfer vector with insert ESSENTIAL GENE X X O SENTIALGENE Digested BacPAK6 viral DNA Recombination Recombinant baculovirus expression vector ESSENTIAL GENE gt Polyhedrin promoter Figure 1 Transfer of a target gene to the baculovirus expression vector by forced recombi nation between a transfer vector and BacPAK6 viral DNA et al 1991 If the second large DNA fragment recircularizes by itself the resulting viral DNA will lack an essential part of the genome and be unable to produce viable viruses However the transfer vector
24. e s Basic Medium Add the Bacfectin DNA mixture dropwise to the medium while gently swirling the dish to mix Incubate at 27 C for 5 hr If positive control was omitted add 1 5 ml of BacPAK Complete Medium to experimental and negative control dishes Incubate at 27 C If positive control was included add 48 ul of X gluc 25 mg ml in DMSO to 4 ml of BacPAK Complete Medium final concentration 300 ug ml Add 1 5 ml of BacPAK Complete Medium X gluc to the negative and positive control dishes Add 1 5 ml of BacPAK Complete Medium to the experimental dish Incubate at 27 C Blue color will be visible in the positive control dish 60 72 hr after adding the Bacfectin DNA mixture The color indicates successful cotransfection and generation of recombinant viruses ex pressing GUS The negative control dish should not change color 72 hr after addition of the Bacfectin DNA mixture to the cells transfer the medium which contains viruses produced by the transfected cells to a sterile container and store at 4 C Optional To obtain more virus add a fresh aliquot 1 5 ml of BacPAK Complete Medium to each dish Incubate at 27 C for another 2 days and harvest the medium as above CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 20 Version PR95847 5 10 11 12 13 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued Use freshly a
25. e agarose in H O previously autoclaved and cool to 37 C Prewarm 10 ml of BacPAK Complete Medium to 37 C 8 Add 24 ul of X gal 25 mg ml in DMF per ml to the prewarmed BacPAK Complete medium 240 ul 10 ml or final concentration 12 ul ml medium when mixed with 2 agarose 9 Remove the virus inoculum from the cells by tilting the dish and aspirating from the edge Proceed immediately to step 10 10 Add warm BacPAK Complete Medium to the agarose and mix this makes a 1 agarose solution which is used to overlay the infected cell monolayer to prevent virus progeny from spreading to other areas of the dish Note Water baths are a major source of microbial contamination therefore dry off the containers and flame the necks before mixing the agarose and medium 11 Gently add 1 5 ml of the agarose overlay to each dish Note Allow the agarose to run down the side of the dish taking care not to disturb the cells 12 When the agarose overlay has set add 1 5 ml of BacPAK Complete Medium to each dish 13 Place dishes in a plastic storage box with a moist paper towel as described in the note to Step 2 incubate dishes at 27 C for 4 5 days Note Stain for virus plaques half the dishes will be stained with neutral red only and half with neutral red and X gal 14 Dilute neutral red to 0 03 with PBS 1 ml of 0 33 w v neutral red stock 10 ml of PBS Add 1 mlofthe 0 03 neutral red solution to each of the 10 dishes Incubate
26. ection with the parental BacPAK6 virus which expresses B galactosidase to high levels and a mock infected control With these controls you can find protein or RNA present in recombinant virus infected cells but not in uninfected cells or in cells infected with wild type or parental viruses TABLE II GUIDELINES FOR PREPARING CELLS FOR ANALYSIS OF TARGET GENE PRODUCTION Number of cells to be seeded Virus Volume Dish flask 2hr overnight Inoculum of Medium 35 mm dish 1 5 1 0 x 106 0 1 0 5 ml 1 5 2 0 ml 60 mm dish 25 2 0 x 108 0 4 1 0 ml 3 0 5 0 ml 150 mm dish 15 0 10 0x 108 2 0 6 0 ml 20 30 ml 25 cm flask 2 0 1 5 x 106 0 4 1 0 ml 3 0 5 0 ml 75 cm flask 6 0 4 0 x 108 0 1 3 0 ml 10 15 ml 150 cm flask 12 0 8 0 x 108 2 0 6 0 ml 20 30 ml When infecting cells for protein production the objectis to get all the cells infected synchronously Therefore a high M O I multiplicity of infection is used Initially an M O I of 10 is recommended but you may also want to try M O I s of 5 and 20 Most proteins expressed from the polyhedrin promoter reach their maximum levels somewhere between 24 hr and 60 hr post infection the best time to harvest depends on the nature of the target protein XI Large scale Target Protein Production The preferred method for producing target protein is to infect cells grown in monolayer culture since it is easier to achieve synchronous infections in monolayer cultures than in suspension cultures However if y
27. ector A Generating a Recombinant Virus Vector DNA e g pBacPAK8 or pBacPAK9 containing the target gene is transfected into Spodoptera frugiperda cells along with 250346 0 BacPAK6 Viral DNA n vivo homologous recombination between the plasmid and viral DNA rescues the viral DNA and transfers the target gene to the viral genome Kitts amp Possee 1993 Kitts 1996 You may use pBacPAK8 GUS as a positive control for the cotransfection step This transfer vector has the E coli B glucuronidase GUS gene cloned downstream of its polyhedrin promoter Recombination of pBacPAK8 GUS with BacPAK6 DNA digest generates recombinant viruses that express B glucuronidase Expression of GUS can be detected by genera tion of a blue color from the chromogenic GUS substrate X Gluc 1 Remove an aliquot of exponentially growing Sf21 cells and dilute with prewarmed 27 C BacPAK Complete Medium to make a 6 ml cell suspension at a concentration of 7 x 10 cells ml 2 Add 1 5 ml of cell suspension approximately 1 x 106 cells to 2 or 3 35 mm tissue culture dishes and rock to distribute the cells Place in a plastic storage box with a moist paper towel and incubate at 27 C for 1 2 hr 3 Remove the medium from the cells and add 2 ml of BacPAK Grace s Basic Medium Swirl gently remove the medium again and add 2 ml of BacPAK Grace s Basic Medium Incubate at room temperature for 10 30 min while the Bacfectin DNA mixture is prepared as described i
28. ed for use in the Polymerase Chain Reaction PCR covered by patents owned by Hoffmann La Roche and F Hoffmann La Roche Ltd Roche No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as CLONTECH Laboratories Inc when used in conjunction with an authorized thermal cycler or is available from Perkin Elmer Corporation Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at the Perkin Elmer Corporation 850 Lincoln Centre Drive Foster City CA 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda CA 94501 SeaPlaque is a registered trademark of FMC Corp BacPAK CHROMA SPIN BacPAK6 pBacPAK8 and pBacPAK9 are trademarks of CLONTECH Laboratories Inc NucleoBond is a registered trademark of Macherey Nagel GmbH and Co 1999 CLONTECH Laboratories Inc CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 Version PR95847 BacPAK Baculovirus Expression System User Manual Appendix B License Agreement for Baculovirus Expression Vector System Taken verbatim from Texas Agriculture Experiment Station TAES form 2 13 90 agmts memoagmt bevs1189 mos
29. em may result in significant numbers of dead cells Nunc scrapers are acceptable but the best ones are made by attaching a piece of silicon rubber tubing to a bent glass rod Alternatively wash cells using a stream of medium from a pipette Sf21 cells attach less strongly to glass and passaging them is easier if you use glass tissue culture flasks 5 Disperse the cells by gently pipeting up and down 3 4 times 6 Transfer the cell suspension to a 150 cm flask containing 30 ml of prewarmed BacPAK Complete Medium 7 Incubate at 27 C until the cells are barely confluent 3 5 days 8 Examine monolayers to check that the cells are healthy and ready for passaging The monolayer should be 80 90 confluent Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 11 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued 9 Remove the old medium and any floating cells If the cells are mainly detached omit this step 10 Add 10 ml of prewarmed BacPAK Complete Medium 11 Genily dislodge the cells using a sterile scraper 12 Disperse the cells by gently pipeting up and down 3 4 times 13 Add 0 3 ml of cell suspension to 0 3 ml of 0 08 w v trypan blue in PBS Count cells with a hemocytometer dead cells take up the blue stain Determine the concentration and proportion of viable cells Note After careful harvesting healthy monolayer cells from plastic flasks
30. ercial use of this system or its components may require additional licenses from the patent holders For additional informa tion please refer to Appendix B Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 3 BacPAK Baculovirus Expression System User Manual l Introduction Baculovirus gene expression is a popular method for producing large guantities of recombinant proteins in insect host cells In most cases posttranslational processing of eukaryotic proteins expressed in insect cells is similar to protein processing in mammalian cells As a result insect cell processed proteins have comparable biological activities and immunological reactivities to proteins expressed in mamma lian cells Protein yields from baculovirus systems are higher and costs are significantly lower than in mammalian expression systems The baculovirus ex pression system can express genes from bacteria viruses plants and mammals at levels from 1 500 mg liter most proteins are expressed in the 10 100 mg liter range although making predictions is difficult The baculovirus most commonly used to express foreign proteins is Autographa californica nuclear polyhedrosis virus AcMNPV Luckow 1991 Vlak amp Keus 1990 Bishop 8 Possee 1990 Miller 1988 Luckow amp Summers 1988 O Reilly et al 1992 ACMNPV can be propagated in certain insect cell lines the virus enters the cells and replication begins app
31. he culture dishes during the incubation period place the dishes in a plastic storage box that has a tight fitting lid place a folded moist paper towel inside the box next to the dishes Seeding dishes with a volume of cell suspension less than 1 5 ml will result in an uneven distribution of cells over the dish The volume added to each dish should be between 1 5 and 2 5 ml 3 Make serial 1 10 dilutions of the BacPAK6 Virus Stock provided in the kit in BacPAK Complete Medium to give final dilutions of 105 and 10 4 Inspect the dishes to ensure that the cells have attached to form an even monolayer of about 70 80 confluency Aspirate the medium from the cells using a sterile pasteur pipette or pipette tip 5 Gently add 100 ul of the virus inoculum to the center of the dish taking care not to displace any cells Infect 4 dishes with the 10 and 4 with the 10 dilution of BacPAK6 Plate 100 ul of the dilution medium onto the remaining two dishes These dishes will be useful for comparing with the infected dishes and they provide a control that will reveal any contamination in the reagents Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 15 BacPAK Baculovirus Expression System User Manual VI Plague Assay Method continued 6 Incubate at room temperature for 1 hr on a level surface to allow the virus to infect the cells 7 During this incubation melt 10 ml of 2 agarose 290 SeaPlagu
32. ions You may culture them as monolayers or in suspension cells can be freely transferred between the two culture types To maintain consistency do not passage cells indefinitely After 20 30 passages replace a culture with fresh cells from liquid nitrogen To prevent contamination work with media and uninfected cells in a vertical laminar flow hood using sterile technique Keep this hood free of virus to avoid accidental infection of the stock cultures ideally use another hood for all virus work Virus infections can be performed on the bench following good microbiological practice unless the recombinant virus carries a potentially harmful or infectious gene Although baculoviruses have a restricted host range treat recombinant baculoviruses as potential biohaz ards All virus contaminated materials including fluids must be autoclaved or disinfected with 5 bleach or a chemical disinfectant before disposal B Culture Media You may propagate Sf21 cells in BacPAK Complete Medium 8090 1 which is fully supplemented You may also use serum free media such as Grace s Basic Medium 8091 1 for assaying or purifying secreted proteins Insect cell medium does not contain pH indicators and is pale yellow The pH of the medium is about 6 2 and it will gradually rise as the cells grow however pH will usually not exceed 6 4 BacPAK Complete Medium contains TNM FH medium Grace s Basic Me dium Grace 1962 with yeastolate lactalbu
33. ith PBS 1 5 ml of 0 33 w v neutral red stock 15 mlofPBS Add 1 mlofthe 0 03 neutral red solution to each of the 14 dishes Incubate at 27 C for 2 3 hr 14 Aspirate off the stain invert the dishes and leave them in the dark at room temperature overnight to allow the plaques to clear 15 Inspect dishes for viral plaques Find dishes on which the diluted cotransfection supernatant produced only a few plaques and mark well isolated ones by circling them with a pen on the outside bottom of the dish Examine under a microscope to ensure that they contain virus infected unstained cells and are not just holes in the cell monolayer Positive control cotransfection In the presence of X gluc recombi nant viruses expressing GUS will give rise to blue plaques that vary in size Small underdeveloped plaques may appear clear but will often become blue on longer incubation 16 Prepare a sterile microcentrifuge tube containing 0 5 ml of BacPAK Complete Medium for each well isolated plaque that you have identified 17 Pick the marked plaques by pushing the tip of a sterile Pasteur pipette through the agarose overlay into the plaque and gently sucking a plug of agarose into the pipette tip Wash the agarose plug into the microcentrifuge tube Vortex and store at 4 C overnight to allow viruses to diffuse out of the agarose This is called a plaque pick Note Nearly all plaques produced from a BacPAK6 cotransfection are recombinant
34. l plaques Therefore developing a good plaque assay technique before working with recombinant viruses is advisable Insert target gene into transfer vector Section VII and prepare plasmid DNA e Produce a recombinant virus by cotransfecting Sf21 cells with BacPAK6 viral DNA and the transfer vector target gene clone Section VIII A e Perform plaque assays on the cotransfection supernatant to obtain indi vidual viral plaques Section VIII C e Test the putative recombinant viruses to confirm that they have incorpo rated the target gene and or express the target protein Section IX A B e Amplify recombinant viruses to obtain working stocks Section IX C D e Titer amplified virus stock Section IX E e Perform small scale infections to characterize gene expression and to determine the optimum harvest time and infection ratio that will give maximum protein yield Section X e Scale up produce target protein in large quantities by infecting larger batches of insect cells Section XI CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 8 Version PR95847 BacPAK Baculovirus Expression System User Manual IV Experimental Outline continued Establish Prepare target vector Section VII Sf21 cells e Insert target gene into transfer vector Section V C e Verify correct construct Pa NA e Plasmid Preparation Freeze cells Maintain working l for long term stocks of Sf21 cells BacPAK6 storage Sections V D
35. liquots of cells in liquid nitrogen provides a source of fresh cells to replace the working stocks when they become old 1 Monitor the cells in a 150 cm flask to ensure that they are healthy and growing exponentially When the monolayer reaches about 80 confluency remove the old medium add 5 ml of prewarmed BacPAK Complete Medium gently scrape the cells and disperse the cells by gently pipeting up and down 2 Count the cells and ensure that they are at least 90 viable 3 Adjust cell density to 4 105 cells ml with BacPAK Complete Medium Chill the cells to 4 C 4 Prepare an equal volume of BacPAK Complete Medium containing DMSO at 20 v v Chill to 4 C 5 Label cryogenic vials and put them on ice 6 Add the BacPAK Complete Medium DMSO to the cell suspension and mix Keep on ice 7 Place 1 ml aliquots of cells into each vial and cap tightly 8 If available place vials in a vapor phase chamber of the liquid nitrogen container and freeze cells slowly overnight before placing in the liquid phase Alternatively place vials at 20 C for 1 2 hr and then in a 70 C freezer overnight Transfer to liquid nitrogen as rapidly as possible 9 After a week or two retrieve one vial and test the viability of the stored cells by following the protocol in Section V C CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 14 Version PR95847 BacPAK Baculovirus Expression System User Manual VI Plague Assay
36. min hydrolysate and L glutamine Hink 1970 supplemented with 10 FBS and 50 ug ml gentamycin You may substitute fully supplemented BacPAK Grace s Medium 8091 1 for BacPAK Complete Medium throughout these protocols Alternatively prepare TNM FH medium Hink 1970 and supplement as follows 1 Add 50 ml of fetal bovine serum cell culture grade preferably insect cell tested to a 500 ml bottle of TNM FH medium to give 10 v v FBS 2 If desired add antibiotics e g 50 units of penicillin and 50 ug of streptomycin per ml of medium or 50 ug of gentamycin per ml of medium from a filter sterilized concentrated stock solution Note Antibiotic use is optional but strongly recommended for cotransfections plaque assays and viral infections because these cultures are prone to contamination 3 Store at 4 C CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 10 Version PR95847 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued C Establishing the Sf21 Cell Line 1 Add5 mlof BacPAK Complete Medium to a 25 cm flask warm to 27 C 2 Remove a vial of cells from liguid nitrogen 3 Thaw rapidly by briefly dipping the vial in a 37 C water bath or by rolling the vial in the palm of your hand Keep at room temperature Note Do not continue to warm the cells after they have thawed Heating cells above 28 C will kill them 4 Immerse or thoroughly swab the vi
37. must obtain an accurate titer for a virus stock so that you can optimize subsequent infections to produce maximal yield of recombinant protein or the highest titer of virus The quickest and easiest method for determining titer is to use the BacPAK Baculovirus Rapid Titer Kit K1599 1 With this kit you can obtain a titer in as little as 48 hours Alternatively you may perform a plaque assay Section VI A In this case dilute amplified virus stock to 104 10 and 10 and infect 2 or 3 dishes of cells for each dilution Use 100 nl of virus dilution per plate Plate out an appropriate dilution of BacPAK6 virus and 100 ul of medium as controls Follow the procedure in Section VI A modifying volumes to match the total number of dishes being used After the appropriate incubation time count the plaques on the plates with reasonable numbers of plaques and calculate the virus titer as explained in Section VI B CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 26 Version PR95847 BacPAK Baculovirus Expression System User Manual X Characterizing Recombinant Gene Expression Before producing the target protein on a large scale characterize gene expression from the recombinantvirus and determine the time course of protein production You can infect cell monolayers of various sizes Table Il to obtain protein or RNA for analysis Always include an infection with wild type virus ACMNPV C6 Possee 1986 and or an inf
38. n the following steps Note A component in serum inhibits transfection this washing step is necessary to replace normal medium with serum free medium before adding the Bacfectin DNA mixture to the cells 4 Dilute the plasmid DNA to 100 ng ul with TE buffer Protocol lt 1 www clontech com CLONTECH Laboratories Inc Version PR95847 19 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued Make the following additions to two or three sterile microfuge tubes optional Tube 1 Tube 2 Tube 3 Experiment Control Control Sterile HO 86 ul 91 ul 86 ul Plasmid DNA 100 ng ul 5 ul 5 ul pBacPAK8 GUS 100 ng ul 5 ul BacPAK6 viral DNA 5 ul 5 ul 85036 digest Final Volume 96 ul 96 ul 96 ul Notes Transfecting the cells with plasmid DNA alone provides a control that will reveal any contamination in the reagents Baculovirus DNA is large and easily damaged by shearing and BacPAK6 DNA will lose its infectivity if it is damaged Therefore the viral DNA should be handled with care throughout these procedures for example to mix solutions containing BacPAK6 DNA gently flick the tube rather than vortexing it Add 4 wl of the Bacfectin to the DNAs and mix gently Incubate at room temperature for 15 min to allow the transfection reagent to form complexes with the DNA Meanwhile remove the medium from the cell monolayers and add 1 5 ml of BacPAK Grac
39. nce from the publishing journal Preprints should be sent to the attention of the Coordinator of Research Development for Industrial Relations Texas Agricultural Experiment Station Texas A amp M University College Station Texas 77843 2162 4 Recipient and those who report to the Recipient are aware of the proprietary interests involved herein and commit to honoring the terms and conditions of this Agreement 5 Recipient accepts the biological material with the knowledge that it is experimental biological material and that it is provided by TAES without warranty of any sort express or implied Recipient agrees to comply with all applicable governmental regulations for the handling thereof Recipient shall hold TAES harmless from any damages which may be alleged to result in connection with the use and possession of the requested materials as provided under this Agreement subject to any relevant state or federal government limitations 6 This Agreement and Recipient s right to use the biological material become effective upon breaking the seal of the package containing the biological material and automati cally terminate if Recipient fails to comply with any provision of this Agreement 7 TAES retains ownership and all rights to biological material not expressly granted and nothing in this Agreements constitutes a waiver of TAES rights under U S Federal State or Patent Law Note These restrictions do not apply to information or te
40. nd are gt 90 viable The density at which the 2 5 x 10 pfu ml cells are seeded for the plaque assay is also critical Solution Be sure to cool agarose to 37 429C before use Try higher dilutions Seed dishes with fewer cells Seed dishes with more cells Be sure to aspirate all of the virus inoculum before add ing the 1 agarose overlay Avoid touching the cell layer with pipettes and tips and make additions gently CLONTECH Laboratories Inc 17 Cause Agarose overlay may have been too hot Virus inoculum may have been too high resulting in complete lysis of the cells Cells may have been seeded too densely Cells may have been seeded too sparsely There may have been some liquid under the agarose overlay Cells may have been disturbed by addition of virus inoculum or the agarose overlay www clontech com Problem Cells are dead whole plate is uniformly red Plagues are very small or invisible Plagues are large but diffuse Plagues appear smeared Cell monolayer contains holes Protocol lt 1 Version PR95847 BacPAK Baculovirus Expression System User Manual VII Construction of a Recombinant Transfer Vector A Tailoring the Insert 1 If using the vectors provided in the kit the target gene must have its own ATG initiation codon which should be the first ATG in the insert 2 The coding seguence must end with a translation termination codon
41. on inhibited by serum or components in the media Viral DNA or Bacfectin damaged by freezing www clontech com Problem Medium on trans fected cells turns cloudy Few or no viral plagues recovered from cotransfection Control transfection generates virus ex pressing GUS but no virus produced in the experimental cotransfection Control transfection does not generate virus expressing GUS medium does not turn blue with X Gluc Protocol lt 1 Version PR95847 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued Isolating Recombinant Viruses Most viruses in the cotransfection supernatant will be recombinant so you can use it as the primary stock of recombinant virus This stock can be amplified titered Section IX and used to express protein Sections X amp XI However it will contain a mixture of viruses and its composition may change with repeated passage resulting in altered expression This short cut can be used to quickly produce a few batches of protein If many batches of protein are to be produced a clonal stock of virus should be produced to ensure consistency You obtain a pure clone of a recombinant virus by diluting the cotransfection supernatant containing progeny viruses and doing a plaque assay to produce individual plaques Optional The positive control cotransfection can be assayed to determine the yield and proportion of
42. ou need more target protein than can practically be produced in a series of 150 mm dishes or 150 cm flasks you may want to use suspension cultures To prepare suspen sion cultures for large scale protein production seed 100 500 ml suspension cultures with 2 x10 Sf21 cells ml and infect them by adding the requisite volume of virus stock when they reach 1 x 105 cells ml To achieve maximal protein expression use BacPAK Complete Medium or other high quality medium and fetal bovine serum and log phase Sf21 cells that are at least 98 viable Protocol PT1260 1 www clontech com CLONTECH Laboratories Inc Version PR95847 27 BacPAK Baculovirus Expression System User Manual XII References Ayres M D Howard S C Kuzio J Lopez Ferber M amp Possee R D 1994 The complete DNA seguence of Autographa californica nuclear polyhedrosis virus Virology 202 586 605 Bishop D H L amp Possee R D 1990 Baculovirus expression vectors Advances Gene Technol 1 55 72 Grace T D C 1962 Establishment of four strains of cells from insect tissues grown in vitro Nature 195 788 789 Hink W 1970 Established insect cell line from the cabbage looper Trichoplusia ni Nature 226 466 467 King L A amp Possee R D 1992 The Baculovirus Expression System A Laboratory Guide Chapman amp Hall NY Kitts P A 1996 Construction of baculovirus recombinants Cytotechnology 20 111
43. recombinant viruses expressing GUS In this case X gluc is added to the overlay in a plaque assay of the control cotransfection supernatant Steps 7 amp 11 1 Seed fourteen 35 mm dishes with 1 x 105 Sf21 cells in 1 5 ml of medium and incubate at 27 C for 1 4 hr 2 Make serial dilutions of the cotransfection supernatant 100 ul into 900 ul of BacPAK Complete Medium to give final dilutions of 101 102 and 10 3 To provide a positive control dilute the BacPAK6 virus stock provided so that 100 ul will produce 10 30 plaques on adish this will be a dilution between 10 and 10 4 Inspectthe dishes from Step 1 above to ensure that the cells have attached to form an even monolayer of about 70 80 confluency Aspirate the medium from the cells using a sterile pasteur pipette or pipette tip 5 Infect4 dishes eachwith the 10 102 and 10 dilutions of the cotransfection supernatant gently add 100 ul of the appropriate virus inoculum to the center of the dish take care not to displace any cells For controls infect one dish with 100 ul of the appropriate BacPAK6 virus dilution and place 100 ul ofthe dilution medium on the remaining dish These controls will be useful for comparison with the infected dishes the negative control will reveal contamination in the reagents 6 Incubate at room temperature for 1 hr on a level surface to allow the virus to infect the cells 7 During this incubation melt 12 ml of 2 SeaPlague agaro
44. roximately 6 hours post infection h p i At approximately 20 48 h p i transcription of nearly all genes ceases The viral polyhedrin and p10 genes however are transcribed at high rates The polyhedrin protein is essential for propagation of the virus in its natural habitat however in cell culture polyhedrin is not needed and its coding sequence can be replaced with a sequence for a target protein Hence the powerful polyhedrin promoter can drive high level transcription of the insert resulting in expression of a recombinant protein that can account for over 30 of total cellular protein The large 134 kb size of the AcMNPV genome Ayres et al 1994 makes direct manipulation of it difficult so recombinant baculovirus expression vectors are constructed in two steps Figure 1 First a target gene is cloned into a modified polyhedrin locus contained in a relatively small transfer vector lt 10 kb The polyhedrin coding sequence has been deleted and replaced with a multiple cloning site MCS A target gene is inserted into this MCS between the polyhedrin promoter and polyadenylation signals Transfer vectors also contain a plasmid origin of replication and an antibiotic resistance gene for propagation in E coli but they are unable to replicate in insect cells In the second step the transfer vector and a viral expression vector are cotransfected into insect cells Double recombination between viral sequences in the transfer vector an
45. se in H O previously autoclaved and cool to 37 C Prewarm 12 ml of BacPAK Complete Medium to 37 C Note To assay for recombinant viruses in the control cotransfection use BacPAK Complete Medium containing 300 ug ml X Gluc Final concentration in agarose overlay is 150 ug ml CLONTECH Laboratories Inc www clontech com Protocol PT1260 1 22 Version PR95847 6 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued 8 Remove the virus inoculum from the cells by tilting the dish and aspirating from the edge 9 Addwarm BacPAK Complete Medium to the agarose and mix this makes a 1 agarose solution which is used to overlay the infected cell monolayer to prevent virus progeny from spreading to other areas of the dish Note Water baths are a major source of microbial contamination therefore dry off the containers and flame the necks before mixing the agarose and medium 10 Gently add 1 5 ml of the agarose overlay to each dish Note Allow the agarose to run down the side of the dish taking care not to disturb the cells 11 When the agarose overlay has set add 1 5 ml of BacPAK Complete Medium to each dish Note To assay for recombinant viruses in the control cotransfection use BacPAK Complete Medium containing 150 ug ml X gluc 12 Place dishes in aplastic storage box with a moist paper towel incubate dishes at 27 C for 4 5 5 13 Dilute neutral red to 0 03 w
46. utoclaved 0 pipette tips etc Sterilize plasmid DNA by ethanol precipitation To rescue pass medium through a 0 2 uM sterile filter and plaque assay the filtrate You may add gentamycin or pen strep to the medium Practice assay Make sure you use healthy cells Carefully compare with plates with medium only Plate the cotransfection supernatant at higher dilutions such as 107 amp 10 Use undiluted cotrans fection medium in plaque assay OR use medium har vested from cotransfection after 4 5 days Also try a control cotransfection using pBacPAK8 GUS Check DNA concentration of experimental transfer vector Clean your plasmid prep on a CHROMA SPIN 400 column Handle viral DNA gently do not vortex Wash soak and wash cells in medium that is protein and serum free Fresh batches of Bacfectin and BacPAK6 viral DNA CLONTECH Laboratories Inc 21 Solution B Troubleshooting Guide Cause Microbial contamination due to contaminated materials or poor sterile technique Poor plaque assay Plaques on control plates will also be small or invisible Dilutions were inappropriate for titer of virus High virus titer will produce confluent plaques that may be mistaken for no plaques Low transfection efficiency Too much or too little plasmid DNA was used Experimental transfer vector DNA contains impurities that inhibit transfection Viral DNA damaged by shearing Transfecti
47. xpression Large scale Target Protein Production Version PR95847 VI VII VIII XI CLONTECH Laboratories Inc www clontech com Protocol lt 1 2 BacPAK Baculovirus Expression System User Manual Table of Contents continued XII References 28 XIII Related Products 29 Appendix A Vector Maps amp Multiple Cloning Site Sequences 30 Appendix B License Agreement for Baculovirus Expression Vector System 33 List of Figures Figure 1 Target gene transfer to baculovirus expression vector 5 Figure 2 Schematic diagram outlining BacPAK procedure 9 Figure 3 Map of pBacPAK8 Transfer Vector 30 Figure 4 Seguences in and around the pBacPAK8 MCS 30 Figure 5 Map of pBacPAK9 Transfer Vector 31 Figure 6 Seguences in and around the pBacPAK9 MCS 31 Figure 7 Map of pBacPAK8 GUS Transfer Vector 32 List of Tables Table Guidelines for seeding densities 12 Table ll Guidelines for preparing cells for analysis of gene production 27 Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use CLONTECH products may not be resold modified for resale or used to manufacture commercial products without written approval of CLONTECH License Agreements The viruses and transfer vectors in the BacPAK Baculovirus Expression System are covered by various patents This system is licensed for use in research only any comm

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