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Guide to Baculovirus Expression Vector Systems (BEVS) …

Instruction ManualGuide to BaculovirusExpression Vector Systems (BEVS) and Insect Cell CultureTechniquesINSIDE FRONT of Baculovirology .. 1 Baculoviruses as Expression Vectors .. 1 Advantages of BEVS 2 Generating a Recombinant Virus by Homologous Recombination ..3 Generating a Recombinant Virus by Site-Specific Transposition ..3 Insect Cell Culture for Culturing Host 6 Protocol 1:Subculturing Monolayer 6 Protocol 2:Adapting Monolayer Cells to Suspension Culture .. 6 Protocol 3: Maintaining Suspension Cultures .. 7 Protocol 4: Adapting Cultures to Serum-Free Medium .. 8 Protocol 5: Preparing a Master Cell Seed Stock .. for Generating a Recombinant 10 Protocol 6:Isolation of Bacmid DNA for BAC-TO-BAC Baculovirus Expression Systemwith the CONCERT High Purity Plasmid Purification system .

After the OV is ingested by insect larvae, the crystalline polyhedrin matrix is degraded in the alkaline mid-gut of the ... Two of the most common isolates used in foreign gene expression are Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) ... or polyhedral inclusion bodies (PIBs)—are formed, and cell lysis begins. Between 24

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Transcription of Guide to Baculovirus Expression Vector Systems (BEVS) …

1 Instruction ManualGuide to BaculovirusExpression Vector Systems (BEVS) and Insect Cell CultureTechniquesINSIDE FRONT of Baculovirology .. 1 Baculoviruses as Expression Vectors .. 1 Advantages of BEVS 2 Generating a Recombinant Virus by Homologous Recombination ..3 Generating a Recombinant Virus by Site-Specific Transposition ..3 Insect Cell Culture for Culturing Host 6 Protocol 1:Subculturing Monolayer 6 Protocol 2:Adapting Monolayer Cells to Suspension Culture .. 6 Protocol 3: Maintaining Suspension Cultures .. 7 Protocol 4: Adapting Cultures to Serum-Free Medium .. 8 Protocol 5: Preparing a Master Cell Seed Stock .. for Generating a Recombinant 10 Protocol 6:Isolation of Bacmid DNA for BAC-TO-BAC Baculovirus Expression Systemwith the CONCERT High Purity Plasmid Purification system .

2 10 Protocol 7:Cationic Liposome-Mediated Transfection Using CELLFECTIN Reagent .. 11 Protocol 8:Virus Plaque Assay .. for Purifying and Producing Recombinant AcNPV and Protein .. 14 Protocol 9:Plaque Purification of Recombinant Viral Clones .. 14 Protocol 10:Amplifying the Virus Stock .. 14 Protocol 11:Identifying Plaques by Neutral Red Staining .. 14 Protocol 12:Optimizing Virus Stock Production .. 15 Protocol 13:Harvesting the Virus .. 15 Protocol 14:Concentrating the Virus .. 15 Protocol 15:Storing the Virus .. 16 Protocol 16:Optimizing Heterologous Protein Recombinant Secreted Proteins ..17 Purifying Intracellular 22 Appendix A: Applications Data for Insect Cell Lines Grown in Serum-Free :1 In vivobaculovirus infection and replication.

3 22 Generating a recombinant Baculovirus by homologous Generation of recombinant baculoviruses and gene Expression with the BAC-TO-BACexpression system ..4 Table of ContentsiiTables:1 Insect cell lines commonly used in BEVS applications ..42 Insect cell culture media commonly used in BEVS applications ..43 Effects of serum on cell Useful medium volumes ..75 Troubleshooting virus plaque assays ..136 Recommended maximum infection densities for the production of rAcNPV or recombinant Maximum cell densities in small-scale suspension culture ..228 -galactosidase Expression in small-scale suspension culture ..239 Pilot-scale recombinant protein Expression in cells rAcNPV titers in small-scale suspension culture.

4 23 BAC-TO-BAC , CELLFECTIN , DH10 BAC , pFASTBAC 1, EXPRESS-FIVE , CONCERT ,TECH-LINESM, and the Life Technologies logo are marks of Life Technologies, BAC-TO-BACB aculovirus Expression system is sold under patent license for research purposes only, and no license for commercial use is for commercial manufacture or use should be directed to the Officer of the Director, Mail Zone 02A, Monsanto Corporate Research, 800 N. Lindbergh, St. Louis, MO is a trademark of Invitrogen is a registered trademark of BASF is a registered trademark of Becton Dickinson & polypeptides gp41 and gp74. Multiple virions areproduced and surrounded by a crystalline polyhedramatrix.

5 The virus particles produced in the nucleus are embedded within the polyhedrin gene product and acarbohydrate-rich 1 summarizes how baculoviruses infect cells andare transmitted in vivovertically and horizontally. During thelytic cycle, enveloped and budded virions are virions promote horizontal transmission of the infec-tion throughout the tissue in an in vivoinfection of a wormlarvae, or throughout the cell culture in an in vitroover- Expression system . In vitro,this cycle is exploited to bothgenerate virus stocks and establish a fully developed infec-tion from subsaturating primary virus inocula. During theoccluded cycle, virions packaged in the PIBs are vivo, these virions promote vertical transmission of thevirus from insect host to insect host.

6 In vitro,a polyhedringene modified to express a recombinant gene product inplace of the PIBs is used. Biochemically, the essential differ-ence between the lytic and occluded cycles is the inductionof polyhedrin production at the beginning of the very need to be able to distinguish between the initiationof virus production and budding, at approximately 8 to 10 hpost-infection, and the initiation of protein Expression undercontrol of the polyhedrin promoter, at approximately 20 to 24h. By doing so, you will be able to effciently produce high-titer Baculovirus stocks and high-quality recombinantproduct ( ,product that is non-degraded and free of celldebris).

7 Vertical TransmissionAfter the OV is ingested by insect larvae, the crystallinepolyhedrin matrix is degraded in the alkaline mid-gut of theinsect. Embedded virions are released and fuse to micro-villar epithelial cells. Infected cells release EV from thebasement membrane side of the mid-gut cell into thehemolymph TransmissionEV enters the insect hemocoel and immediately spreadsthroughout the insect s open circulatory system , infectingmany cell types. Within 10 viral generations, the insect diesand the OV, produced during the very late stage of infection,is released into the as Expression VectorsThe major difference between the naturally occurring invivo infection and the recombinant in vitroinfection is thatthe naturally occurring polyhedrin gene within the wild-typebaculovirus genome is replaced with a recombinant gene orcDNA.

8 These genes are commonly under the control ofpolyhedrin and p10 promoters. In the late phase of infection,the virions are assembled and budded recombinant virionsare released. However, during the very late phase of infec-tion, the inserted heterologous genes are placed under thetranscriptional control of the strong AcNPV polyhedrinpromoter. Thus, recombinant product is expressed in place1 Recombinant baculoviruses are widely used toexpress heterologous genes in cultured insect cellsand insect larvae. For large-scale applications, thebaculovirus Expression Vector system (BEVS) is particularlyadvantageous. Specialized media, transfection reagents,and vectors have been developed in response to recentadvances in insect cell culture and molecular biology following are important choices in designing a system for recombinant protein production: Selecting the Expression Vector , including the style ortype of promoter, that provides best results with therecombinant gene product being expressed.

9 Evaluating insect cell lines, growth media (serum-supplemented or serum-free), and feeding/infectionstrategies that allow for optimal rAcNPV and/or productexpression. Choosing a scalable process of cell culture and decidingon other factors affecting downstream of BaculovirologyBaculoviruses are the most prominent viruses known toaffect the insect population. They are double-stranded,circular, supercoiled DNA molecules in a rod-shaped capsid(1). More than 500 Baculovirus isolates (based on hosts oforigin) have been identified, most of which originated inarthropods, particularly insects of the order Lepidoptera(2,3). Two of the most common isolates used in foreigngene Expression are Autographa californicamultiple nuclearpolyhedrosis virus (AcMNPV) and Bombyx mori(silkworm)nuclear polyhedrosis virus (BmNPV).

10 Wild-type baculoviruses exhibit both lytic and occludedlife cycles that develop independently throughout the threephases of virus replication. The following are characteristicsof the three Phase: In this phase (also known as the virussynthesis phase), the virus prepares the infected cell forviral DNA replication. Steps include attachment, pene-tration, uncoating, early viral gene Expression , and shutoff of host gene Expression . Actual initial viral synthesisoccurs to 6 h after Phase:In this phase (also known as the viral struc-tural phase), late genes that code for replication of viralDNA and assembly of virus are expressed.


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