key: cord-0036103-sy3ri0k9 authors: Fukai, Shuya title: Application of MultiBac System to Large Complexes date: 2016-03-03 journal: Advanced Methods in Structural Biology DOI: 10.1007/978-4-431-56030-2_3 sha: 8490aad5f39c86e31c43cc3eca6090402e895753 doc_id: 36103 cord_uid: sy3ri0k9 Multisubunit protein complexes regulate numerous biologically important processes. Elucidation of their functional mechanisms based on their three-dimensional structures allows us to understand biological events at the molecular level. Crystallography and electron microscopy are powerful tools for analyzing the structures of biological macromolecules. However, both techniques require large-scale preparation of pure and structurally homogenous samples, which is usually challenging for large multisubunit complexes, particularly from eukaryotes. In this chapter, we describe the principles and methods of producing multisubunit complexes in insect cells using the MultiBac system. genes of interests with their upstream ribosome binding sites (i.e., the Shine-Dalgarno sequence) between one promoter in the 5 0 -end and one terminator in the 3 0 -end enables polycistronic expression of proteins. Furthermore, cotransformation with two or three expression plasmids carrying different antibiotic resistance markers can be used for the coexpression. However, many eukaryotic protein complexes have subunits that are difficult to produce in E. coli, possibly owing to problems on transcription, translation, folding, and/or posttranslational modifications (e.g., phosphorylation and glycosylation), resulting in the failure of efficient production in E. coli. An excellent solution to this difficult protein production in E. coli is the use of a baculovirus expression system, which employs an engineered baculovirus genome derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) and appropriate transfer vectors (Fig. 1) . Polyhedrin and p10 are products from the AcNPV genome and highly accumulated in the late stage of infection [1] . Their transcription promoters allow overproduction of proteins in insect cells. Genes of interest are first inserted between one of these strong transcription promoters (polyhedrin or p10 promoter) and a polyadenylation signal (SV40 or HSVtk polyadenylation signal) in the transfer vector and then integrated into the engineered AcNPV genome. The engineered AcNPV genome containing genes of interest is transfected to cells of the caterpillar Spodoptera frugiperda Sf9 or Sf21 cell line. The transfected Sf cells produce viruses that can infect insect cells to overexpress genes of interest. Suspension culture of the transfected Sf cells in shaker flasks is convenient and effective for large-scale protein production. The genes of interest can be integrated to the engineered AcNPV genome in either insect cells or E. coli through the transfer vector. In the first case, both the linearized AcNPV genome containing a lethal deletion and the transfer vector are cotransfected into insect cells, and recombination occurs between the engineered AcNPV genome and the transfer vector. Because the transfer vector can rescue the lethal deletion of the engineered AcNPV genome, viruses that are generated from the genome fused with the transfer vector propagate. However, practically, this strategy might generate nonrecombinant viruses, which could apparently decrease virus titer (infection efficiency) and gene expression level. In such a case, a virus species with higher titer should be isolated from a single plaque by a plaque assay, which may require additional time and effort. This step can be bypassed when genes of interest are integrated to the engineered AcNPV genome in E. coli cells, applying the transposing reaction of the Tn7 transposon [2] . The Tn7 transposon is a relatively large DNA segment (14 kb) from the Tn7 phage, which can be integrated into a specific position called the Tn7 attachment site (attTn7) in the E. coli genome with high frequency. Tn7 transposon encodes a Tn7 transposase complex, which catalyzes the insertion of DNA elements containing the specific sequences Tn7L and Tn7R into the attTn7 site of another DNA molecule. As the transfer vector containing Tn7L and Tn7R is introduced to specific E. coli strains with a copy of the engineered AcNPV genome containing attTn7 and a helper plasmid encoding the Tn7 transposase complex, the transfer vector is transposed to the attTn7 site of the engineered AcNPV genome in the E. coli cells. Because the attTn7 site in the engineered AcNPV genome is located in the middle of lacZα, positive clones (i.e., E. coli cells containing the transposed AcNPV genome) are easily selected by standard blue-white selection in the presence of X-Gal and IPTG. The integrated AcNPV genome can then be isolated from the positive clones by a standard alkaline lysis protocol and used for the subsequent transfection to S. frugiperda cells. In the baculovirus-insect cell system, coinfection by several viruses is one of two options for multigene expression: viruses expressing each subunit of the complex are first prepared and then simultaneously applied to infect S. frugiperda cells for coexpression of the appropriate subunit combination. However, this strategy can only be used under specific conditions because it is difficult to achieve a uniform expression of individual subunits owing to the differences in virus titer and gene expression level. Differences in the expression level among individual subunits may result in the failure of homogeneous complex formation. Another versatile option is multigene incorporation to a single AcNPV genome to generate a single species of virus that can produce multisubunit proteins. The MultiBac expression system was designed and developed for this purpose by Berger and colleagues [3] [4] [5] . Multigene incorporation to a single AcNPV genome requires a transfer vector containing a set of genes of interest. The MultiBac system enables two distinct methods for easier construction of the transfer vector for this purpose: one is the use of a multiplication module (Fig. 2 ) and the other is recombinase-mediated vector fusion (Fig. 3 ). Transfer vectors for the "first-and second-generation (old)" MultiBac systems (i.e., pKL, pFL, pUCDM, pSPL, pFBDM, and pKDM) [3, 5] (Fig. 2a) have two multicloning sites, flanked by the restriction sites PmeI and AvrII. In addition, between these two multicloning sites, there is the multiplication module M to be digested by the restriction enzymes BstZ17I and SpeI, which generate cohesive ends compatible with those generated from PmeI and AvrII, respectively. Therefore, a multigene expression cassette withdrawn from one transfer vector by PmeI-AvrII digestion can easily be incorporated into the BstZ17I-SpeI-digested multiplication module M in another transfer vector (Fig. 3a) . Iteration of the incorporation using the multiplication module can multiply expression cassettes in the transfer vector. Similarly, in the "third- (Fig. 3b) . Multiplication of an expression cassette using the multiplication module should enable the incorporation of unlimited numbers of genes to the transfer vector in principle. However, practically, iterative incorporation of the expression cassette generates DNA plasmids that are too large to handle. To address this issue, for the MultiBac system, an additional concept is applied; that is, a number of transfer vectors can be fused by recombination to generate a multigene transfer vector that contains larger numbers of genes ( Fig. 4) . Transfer vectors used for the MultiBac system are classified into two groups: "acceptors" and "donors" (Fig. 2 ). Both acceptors and donors have a short imperfect inverted repeat (LoxP) for the recombination reaction catalyzed by Cre recombinase [6] . Only acceptors contain Tn7L and Tn7R for integration to the engineered AcNPV genome. Acceptors have a ColE1 DNA replication origin and can propagate in standard E. coli cloning strains, whereas donors have an R6Kγ DNA replication origin and require a pir gene product for their propagation. Therefore, donors can be retained in pir-negative E. coli strains only when they are fused with acceptors. Propagation of donors in pir-negative E. coli strains indicates that the donors are appropriately fused with acceptors. This is an important feature because E. coli cells can retain two or more independent (i.e., unfused) plasmids with distinct antibiotic markers simultaneously in the presence of the antibiotics. One acceptor can be fused with one or two donors by in vitro Cre recombinase reaction coli strains with the resultant multigene transfer vector in the presence of the appropriate combination of antibiotics. In the new MultiBac system, the Cre-mediated fusion with three donors can be practically performed in two steps. The MultiBac system also applies new technologies for the engineered AcNPV genome, where genes encoding viral protease and apoptotic activities are deleted to avoid protein degradation and delay lysis of the infected insect cells. Furthermore, in addition to the attTn7 site, the MultiBac AcNPV genome contains the LoxP site, which is useful for additional functionalities. For example, when a yellow fluorescence protein (YFP) gene is integrated to the LoxP site of the MultiBac AcNPV genome, the produced YFP provides information about protein production and virus performance through its fluorescence almost in real time. This YFPintegrated MultiBac AcNPV genome (EMBacY) is included in the new MultiBac system kit. Many of the recent structural studies of biologically important eukaryotic multisubunit complexes utilize the MultiBac expression system for their production [7] [8] [9] (Fig. 4) . All of such complexes were challenging targets of structural studies because of their large size and/or complicated subunit composition. The anaphase-promoting complex (APC/C) is a cell cycle regulator and composed of 13 subunits with a total molecular weight of~1.1 MDa. The gene assembly encoding the complete APC/C was inserted into two MultiBac baculoviruses, one encoding eight subunits and the other five. Coinfection by two viruses enables the production of the entire 1.1 MDa APC/C complex. The purified complex was subjected to electron microscopy analysis, revealing the complicated subunit architecture of this huge complex [9] (Fig. 5) . The transcription mediator controls transcription through its interactions with RNA polymerase and transcriptional activators. The mediator consists of 25 or more subunits with a total molecular weight of~1.2 MDa. The head module of the yeast mediator (seven subunits, Mw 223 kDa) was produced using the MultiBac system and subjected to cryo-EM analysis and X-ray crystallography, revealing the architecture and dynamics at the atomic level [8] . A general transcription factor, TFIID, binds gene promoters and regulates the initiation event of transcription. TFIID is composed of the TATA-box-binding protein (TBP) and 13 TBPassociated factors (TAFs) with a total molecular weight of over 1 MDa. The TFIID core complex (five subunits, Mw 650 kDa) was produced using the MultiBac system and subjected to cryo-EM analysis, revealing its subunit stoichiometry and architecture [7] . For the production of this TFIID core complex, an additional technology was applied for the uniform expression of the individual subunits. Coexpression of the TFIID core subunits by the original MultiBac system showed imbalanced production of the individual subunits, which hampered the purification of the complexes. This imbalanced expression problem was solved by a polyprotein strategy, where a number of proteins are encoded in one large open reading frame (ORF) and generated by proteolysis with a highly specific protease in a manner similar to protein production by RNA viruses such as the coronavirus. The TAF-encoding genes are concatenated into a single ORF, spaced by cleavage sites for a protease NIa from the tobacco etch virus (TEV). The TEV protease gene precedes the TAF genes in the ORF. A cyan fluorescent protein (CFP) gene is also inserted in the 3 0 end of the ORF for checking the expression of all conjoined proteins. These structural studies of large protein complexes including those with molecular weights of over 1 MDa demonstrate that the Three-dimensional structures of large multisubunit complexes analyzed using MultiBac expression system. For APC/C, two subcomplexes (named TPR5 and SC8) were also analyzed besides the entire APC/C MultiBac system is a powerful tool for the overproduction of challenging multisubunit protein complexes. Higher-resolution analysis of complex structures requires more homogeneous samples in terms of size, composition, and posttranslational modification and conformation, which typically require the removal of specific subunits and/or modification of the individual subunits by sitedirected mutations and/or trimming regions that are predicted or experimentally shown to be flexible or disordered. The modular concept of the MultiBac system is highly compatible with this optimization process. It has been announced that the MultiBac system is still under development and that new technologies are planned to be included. This powerful tool for the production of multiprotein complexes will further accelerate the study of the structural biology of challenging targets to elucidate more complex biological processes. 1. Linearize 3-5 μg of vectors (for ten or less SLIC reactions) by either PCR or restriction enzyme digestion and purify them by agarose gel electrophoresis (see Note 2). 2. Set up SLIC reaction (10 μL for each), as shown in Table 1. 3. Incubate the reaction mixture at 50 C for 15-20 min and stop the reaction by placing the mixture on ice. 4. Transform chemically competent E. coli cells with 1 μL of the reaction. Typical E. coli cloning strains (e.g., DH5α) are available for vectors harboring the ColE1 or pBR322 replication origin (i.e., pFL, pKL, pACEBac1, and pACEBac2), whereas a special strain (PirHC or PirLC) is required for vectors harboring the pir replication origin (i.e., pUCDM, pSPL, pIDC, pIDK, and pIDS) (see Table 2 ). Table 2 ) and incubate them at 37 C for 12-15 h. Each colony is picked with a sterilized toothpick, and the toothpick is briefly dipped into a tube containing a PCR reaction mix. The reaction products are separated by agarose gel electrophoresis. We use the 5 0 and 3 0 primers derived from the insert and vector sequences, respectively, to confirm that the insert is integrated into the vector. Typically, four to eight colonies are sufficient to obtain two or more positive clones. 7. Isolate vectors from positive clones using a commercially available mini-prep kit (e.g., Promega Wizard Plus SV Minipreps DNA Purification System) and verify the nucleotide sequences of the insert by DNA sequencing. Promoter and terminator regions should also be verified when vectors are linearized by PCR in Step 1. 8. Store the verified vectors at À20 C until use. Multigenes can be assembled in a single vector by using a multiplication module (Fig. 3) . Genes inserted into the old vectors (i.e., pKL, pFL, pUCDM, and pSPL) can be excised as a cassette by restriction enzyme digestion with PmeI and AvrII. This cassette can be transferred to another vector digested with BstZ17I and SpeI. Similarly, genes inserted into the new vectors (i.e., pACEBac1, pACEBac2, pIDC, pIDK, and pIDS) can be excised by BstXI digestion and either I-CeuI or PI-SceI digestion and transferred to another vector digested with BstXI within either of the donor vectors or acceptor vectors. Step 3.1.6, except that the 5 0 and 3 0 primers derived from the insert are used for colony PCR analysis because the upstream and downstream sequences are the same in the cassettes. 5. Isolate the vector from the positive clone using a commercially available mini-prep kit (e.g., Promega Wizard Plus SV Minipreps DNA Purification System) and store it at À20 C until use. Cre-mediated fusion is also available for generating multigene transfer vectors. In both the new and old systems, it is guaranteed that one acceptor vector can be fused with one or two donor vectors. Furthermore, fusion between one acceptor and three donors is possible in the new system. 1. Set up the Cre reaction (10 μL for each), as shown in Table 3 , and incubate the reaction mixture at 37 C for 30 min to 1 h (see Note 4) . Optionally, the reaction can be stopped by heating at 65 C for 5 min. To avoid the integration of more than one acceptor, the amount of the acceptor vector should be lower than those of the donor vectors. 2. Transform the pir-negative E. coli strain (e.g., DH5α) with 1 μL of the Cre reaction mixture. Cre fusion generates longer vectors, particularly in the old system. Therefore, we use electrocompetent cells for transformation with the Cre-fused vectors. 3. Select positive clones as in Step 3.1.6. Confirm that all genes are present in the vector by PCR analysis after the vector isolation (see Note 5) and store the vector at À20 C until use. Multigene transfer vectors constructed in Steps 3.2 and/or 3.3 are integrated into the engineered AcNPV genome retained in special E. coli strains such as DH10Bac and DH10MultiBac (see Note 6). DH10MultiBac is deficient in protease and chitinase to reduce proteolysis and extend cell viability. For the transformation, we use electrocompetent cells (see Note 7). 1. Prepare electrocompetent DH10Bac or DH10MultiBac cells that retain the engineered AcNPV genome and helper plasmid. (f) Gently suspend the obtained cell pellet in 500 mL of the cold sterilized deionized water from Step (a). (g) Centrifuge the suspension as in Step (d) and remove the supernatant as in Step (e). (h) Repeat Steps (f) and (g). (i) Gently suspend the obtained cell pellet in 10 mL of the cold sterilized 10 % glycerol from Step (a). (j) Repeat Step (g). (k) Gently suspend the cell pellet in 2 mL of the cold sterilized 10 % glycerol from Step (a). (l) Flash-freeze this suspension in 50-200 μL aliquots in liquid N 2 and store them at À80 C until use. 2. Mix 1 μL of 100-500 ng/μL multigene transfer plasmid with 50 μL of electrocompetent cells and incubate it on ice for 5 min. 3. Transfer the cells to an electroporation cuvette and electroporate them using the appropriate electroporation equipment (e.g., MicroPulsor, BioRad). 4. Suspend the cells with 500 μL of LB medium (or richer medium such as SOC) in the cuvette and transfer them to a 1.5 mL tube. 5. Incubate the cells at 37 C with shaking for 6 h. 6. Transfer 10 μL of the culture to 1 mL of LB medium in a new tube. Then, 10 μL of the diluted culture is transferred to 90 μL of LB medium in another new tube. 7. Streak 100 μL of these two diluted cultures on LB plates containing 50 mg/L kanamycin, 7 mg/L gentamicin, 10 mg/L tetracycline, X-Gal (or an equivalent indicator), and IPTG. We supply 50 μL of 2 % X-Gal and 25 μL of 0.2 M IPTG for each antibiotic-containing plate just before streaking the cells. The culture dilution is important for optimal colony separation. 8. After the incubation at 37 C for 2 days, larger white colonies appear if the genes in the transfer plasmid are successfully integrated to the engineered AcNPV genome in E. coli cells. Restreaking on a fresh plate is recommended to confirm the white phenotype. 9. Pick a white colony for each construct and inoculate it to 5 mL of LB medium supplemented with 50 mg/L kanamycin, 7 mg/ L gentamicin, and 10 mg/L tetracycline in a culture tube. 12. Add 300 μL of Solution 2 (0.2 N NaOH, 1 % SDS, see Note 8) and gently mix the solution by inverting the tube upside down several times, followed by incubation at room temperature for 5 min. 13. Slowly add 300 μL of Solution 3 (3 M potassium acetate, pH 5.5, see Note 8) and gently mix the solution by inverting the tube upside down several times, followed by incubation on ice for 5 min. A thick white precipitate of E. coli proteins and genomic DNA appears. 14. Clear the solution by centrifugation at 14,000 Â g for 10 min. During the centrifugation, label another fresh 2 mL tube and add 0.8 mL of 2-propanol to it. 15. Carefully transfer the supernatant to the 2 mL tube containing 2-propanol to avoid contamination of the white precipitate as much as possible and mix it gently by inverting the tube upside down a few times. 16. Place the tube on ice for 10 min and centrifuge it at 14,000 Â g for 10 min at room temperature. The multigene-integrated bacmid is precipitated as a translucent pellet in this step. 17. Remove the supernatant and add 500 μL of 70 % ethanol. 18. Centrifuge the tube at 14,000 Â g for 5 min at 4 C or room temperature . 19. Remove the supernatant as much as possible and air-dry the pellet (the integrated AcNPV genome) in a sterile hood to avoid contamination of microorganisms in the transfected cells. 20. Store the dried pellet at À20 C until use (Note 9). The following steps should be performed in a sterile hood to avoid contamination of insect cell culture: 1. Add 30 μL of sterile pure water to the tube containing the isolated AcNPV genome pellet. 2. Dissolve the pellet by gently tapping the tube and incubate the tube at room temperature for 5-10 min to completely dissolve the pellet. 3. Add 200 μL of an antibiotic-free insect cell medium (e.g., Grace's insect cell medium) to the tube containing the bacmid solution (Tube A). 4. Add 100 μL of an antibiotic-free insect cell medium to another fresh tube (Tube B). 5. Add 8 μL of the transfection reagent, X-Treme GENE HP (Roche), to Tube B. 6. Transfer the mixture containing the transfection reagent in Tube B to Tube A containing the isolated AcNPV genome, followed by incubation at room temperature for 15-30 min. 7. During the incubation, seed 0.5-1.0 million cells to each well of a 6-well tissue culture plate (see Note 10) and add a supplemented insect cell medium [containing antibiotics and/or 4 % fetal bovine serum (FBS)]) with a final volume of 3 mL for each well (Fig. 6 ). 8. Add one-half of the mixture containing the isolated AcNPV genome and transfection reagent dropwise to each well. 1. Prepare 9 mL of cell culture (containing 4 % FBS and antibiotics) at a density of 0.5-1.0 million cells/mL in 125 or 250 mL Erlenmeyer flasks (see Note 12). For virus amplification, we use flasks whose volumes are 10-to 25-fold larger than the culture volume. 2. Inoculate 1 mL of the P1 virus to a 9 mL cell culture for infection. 3. Culture the infected cells at 27 C for 60-72 h with shaking. We recommend the monitoring of cell growth every 12-24 h. We suggest that cell density should be lower than 1.5 million cells/mL to avoid insufficient aeration. When the density is over 1.5 million cells/mL, dilute the culture to a density of 0.5-1.0 million cells/mL, keeping the culture volume at 1/ 10-1/25 of the vessel. 4. Centrifuge the culture for 5 min at 2000 Â g and collect the supernatant (P2 virus). The cell pellet after the centrifugation is used in Step 6. 5. Store the P2 virus at 4 C (or À80 C after flash-freezing in liquid N 2 for long-term storage) protected from light (see Note 13). 6. Check the protein expression using the cell pellet collected by centrifugation (see Note 14). (a) Suspend the cell pellet in 300 μL of an appropriate buffer for tag-affinity beads (e.g., 50 mM Tris-Cl, pH 8.0, containing 150 mM NaCl and 20 mM imidazole for Ni-chelating beads) and transfer it to a 1.5 mL tube. (b) Disrupt the cells by sonication (e.g., Branson Sonifier 450A equipped with a microtip) for 15 s on ice. Take 5 μL as the whole extract for SDS-PAGE analysis (Sample A). (c) Centrifuge the disrupted cell suspension at 20,000 Â g for 15 min at 4 C. During the centrifugation, preequilibrate 15 μL of affinity beads with a sonication buffer in a fresh 1.5 mL tube, following the manufacturer's instruction. (d) Take 5 μL of the supernatant as the soluble extract (Sample B) and pick a very small amount of the resulting pellet with a micropipette tip as the insoluble extract (Sample C) for SDS-PAGE analysis. (e) Transfer the remaining supernatant to the fresh 1.5 mL tube containing pre-equilibrated affinity beads. (f) Incubate the sample for 0.5-1 h at 4 C. (g) Centrifuge the sample at 500 Â g for 3 min and remove the supernatant. (h) Wash the beads with 500-1000 μL of a sonication buffer (or a more stringent buffer of choice) three times by iteration of buffer addition, centrifugation, and buffer removal. Take 5 μL of each wash solution as the wash samples (Samples D-F) for SDS-PAGE analysis. (i) Add 20 μL of an SDS-PAGE loading buffer as the purified sample (Sample G) for SDS-PAGE analysis. (j) Analyze Samples A-G by SDS-PAGE with standard Coomassie brilliant blue and/or immunostaining. For large-scale protein production, we use 2 L Erlenmeyer flasks containing 400-500 mL of cell culture (see Note 15). For example, two flasks are used for 0.8-1 L cell culture. The number of flasks depends on the expression level of your target protein. We use a fresh virus preparation propagated from the stock P2 or P3 virus for large-scale protein production. 1. Inoculate 250 μL of the stock P2 or P3 virus to 25 mL of cell culture (containing antibiotics and 4 % FBS) for infection at a density of 0.5-1 million cells/mL in 0.5 L flask to obtain fresh virus preparations for large-scale expression. 2. Culture the infected cells at 27 C for 60-72 h. 3. Seed cells to 400-500 mL media containing antibiotics and 4 % FBS at a final density of 0.3 million cells/mL for each flask 1 day after Step 1. 4. Add 25 mL of the infected cell culture (from Steps 1-2) to a fresh 400-500 mL cell culture at a density of one to two million cells/mL for each flask (from Step 3). Step 4 at 27 C for 60-72 h (see Note 16). 6. Collect the cells by centrifugation at 2000 Â g. 7. Flash-freeze the cells in liquid N 2 and store them at À80 C until use. 1. PirHC and PirLC are strains required for preparation of donor plasmids (i.e., pIDC, pIDK, pIDS, pUCDM, pSPL). These two strains contain the pir gene in their genomes and can propagate plasmids that have pir replication origins. LC and HC mean low copy and high copy, respectively. PirLC strains are used when inserted genes make the propagation of donor plasmids difficult in PirHC. 2. We favor restriction enzyme digestion for the linearization of vectors for SLIC in order to avoid PCR-associated mutations. 14. Protein expression level may depend on incubation time. You can determine when cells stop proliferation after infection and analyze time-dependent changes in expression level by sampling one million cells every 12 or 24 h after the proliferation arrest. 15. For protein production, High Five cells (derived from the caterpillar Trichoplusia ni) may be more suitable than Sf cells in some cases. 16. Optionally, 1 day after infection, the infected cells are cultured at 20 C for 72 h. This change in culture temperature increases the expression level and/or protein solubility in some cases. Physical analysis of autographa californica nuclear polyhedrosis virus transcripts for polyhedrin and 10,000-molecular-weight protein Tn7: a target site-specific transposon Baculovirus expression system for heterologous multiprotein complexes MultiBac: expanding the research toolbox for multiprotein complexes Protein complex expression by using multigene baculoviral vectors Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination The architecture of human general transcription factor TFIID core complex Architecture of the mediator head module Structural basis for the subunit assembly of the anaphase-promoting complex Tandem recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs for protein complex research TIPS: titerless infected-cells preservation and scale-up This work was supported by grants from MEXT and JST CREST. to the cassette and cannot be removed by gel extraction. To avoid this problem, we recommend using vectors with two different antibiotic resistance makers for the cassette-mediated multiplication (e.g., the cassette from pUCDM is transferred to pFL).4. Although we never tried fusion with more than two donors, an online manual for the new system describes in detail the protocol for fusion with more than two donors. Following such a protocol, the Cre reaction is performed at 30 C for 1-2 h with 500 ng of each donor and~400 ng of one acceptor (http:// www.epigenesys.eu/images/stories/protocols/pdf/20120313 121202_p54.pdf).5. According to the online manual (http://www.epigenesys.eu/ images/stories/protocols/pdf/20120313121202_p54.pdf), restriction analysis using appropriate restriction enzymes is highly recommended for confirming the presence of all genes in the vector.6. We try both DH10Bac and DH10MultiBac and compare them.7. Protocols for chemically competent cells are described in the online manual for the new system (http://www.epigenesys.eu/ images/stories/protocols/pdf/20120313121202_p54.pdf).8. Solutions 1, 2, and 3 are our own preparations, but solutions from commercially available plasmid mini-prep kits can be used for the bacmid isolation.9. We quickly move to Step 3.5 after isolating the integrated AcNPV genome. 10 . A 35 mm culture dish is available for this purpose. However, we recommend a 6-well plate because media in the culture dish seem to evaporate much faster than those in the 6-well plate.11. We quickly move to Step 3.6 after preparing P1 virus.12. We reuse plastic Erlenmeyer flasks supplied by Corning after autoclaving.13. Baculoviruses can be stored in liquid N 2 in the form of baculovirus-infected insect cells (BIICs), which is widely adopted in many laboratories [11] (http://www.epigenesys.eu/ images/stories/protocols/pdf/20120313121202_p54.pdf). This BIIC storage is recommended in terms of space saving in refrigerators and almost no loss of titer. Some viruses lose their transfection activity within a month or less when stored in the form of a virus solution at 4 C, while others retain the activity for more than a 2-3 years.