ExpiSf Expression System - Thermo Fisher Scientific
ExpiSf Expression SystemUSER GUIDEFor superior baculovirus-based protein expression using high-densityExpiSf9 cells in a chemically defined, yeastolate-free mediumCatalog Numbers A38841, A39111, A39112Publication Number MAN0017532Revision A.0For Research Use Only. Not for use in diagnostic procedures.
Manufacturer: Life Technologies Corporation 5781 Van Allen Way Carlsbad, CA 92008The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.Revision history: Pub. No. MAN0017532RevisionDateA.026 April 2018DescriptionNew documentImportant Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept theterms and conditions of all applicable Limited Use Label Licenses.TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. 2018 Thermo Fisher Scientific Inc. All rights reserved.
Contents Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Components of ExpiSf Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf CD Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiFectamine Sf Transfection Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf Enhancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opti-MEM I Reduced Serum Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bac-to-Bac Baculovirus Expression System components . . . . . . . . . . . . . . . . . . . . . . . 88889999Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Guidelines for ExpiSf9 cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Cell handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Growth media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Thaw and establish ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thaw ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subculture ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Passage ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Cryopreserve cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121212121313131314Baculovirus generation: Transfect ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf9 cell transfection at different scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ExpiSf9 cell transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transfect ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151515151617Baculovirus generation: Isolate and amplify P0 viral stock . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Isolate P0 virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19191919ExpiSf Expression System User Guide3
ContentsGuidelines to amplify baculovirus stock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Day 1: Prepare cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Day 0: Infect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Day 3–5: Harvest virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein expression in ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Protein expression at various scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infect ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Day 1: Prepare ExpiSf9 cells for infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Day 0: Infect ExpiSf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Optimize protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202020202121212222222323APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24General cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Baculovirus generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Protein expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 APPENDIX B Generate recombinant bacmid DNA . . . . . . . . . . . . . . . . . . . 29Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Generate pFastBac construct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Cloning considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .E. coli host for transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Analyze transformants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Analyze transformants by PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Long-term storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429292929303030303131Transform into DH10Bac competent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Positive control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prepare LB agar plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prepare for transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Transform cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Verify the phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31313132323233Isolate and analyze recombinant bacmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Isolate recombinant bacmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Analyze recombinant bacmid DNA by PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PCR analysis with pUC/M13 primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Produce the PCR product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PCR product size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34343434343535ExpiSf Expression System User Guide
Contents APPENDIX C Assay for β-glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Assay for β-glucuronidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 1: Prepare Gus test sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 2: Dilute Gus test sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 3: Prepare GUS recombinant protein standard . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 4: Prepare FDGlu Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 5: Set up assay plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Step 6: Calcuate Gus protein concentration in test sample . . . . . . . . . . . . . . . . . . . . . . APPENDIX D Baculovirus titering assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Baculovirus titering assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Titer baculovirus: Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Titer baculovirus: Day 2 (A.M.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3737373738383939394040404141 APPENDIX E Transfect ExpiSf9 cells using adherent-basedtransfection protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Adherent-based transfection protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Transfect cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 APPENDIX F Multiplicity of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Multiplicity of Infection calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 APPENDIX G Vector maps and multiple cloning sites . . . . . . . . . . . . . . . 46 pFastBac 1 vector map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Multiple cloning site in pFastBac 1 vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 pFastBac 1-Gus vector map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 APPENDIX H Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 ExpiSf Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Bac-to-Bac and related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Cloning products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Plasmid purification products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Protein extraction and quantification products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Protein purification products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Nalgene flasks products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52ExpiSf Expression System User Guide5
Contents APPENDIX I Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 APPENDIX J Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566ExpiSf Expression System User Guide
Product informationIMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.Product descriptionThe Gibco ExpiSf Expression System is a high-yield baculovirus-based insectexpression system based on suspension-adapted Sf9 (Spodoptera frugiperda) cells. TheExpiSf Expression System Starter Kit provides cells, culture medium, and reagents toinfect 1 liter of cell culture.Contents and storageThe ExpiSf Expression System contains the following components. For a detailed description see “Components ofExpiSf Expression System“.Contents[1]AmountStorageExpiSf9 Cells (1 107 cell/mL)2 1.5 mLLiquid nitrogen, vapor phase[2]ExpiSf Protein Production Kit:— ExpiSf CD Medium1L ExpiSf Enhancer2 C to 8 C. Protect from light.4 1 mLExpiFectamine Sf Transfection Reagent1 mL2 C to 8 C. Do not freeze.Opti-MEM I Reduced Serum Medium [3]500 mL2 C to 8 C. Protect from light.pFastBac 1 Expression Vector (0.5 µg/ µL) [3,4]10 µg 20 CpFastBac 1-Gus Control Vector (0.2 ng/ µL) ([3,4]4 ng 20 CCompetent cells: 80 C MAX Efficiency DH10Bac CompetentCells[3]5 100 µLpUC19 DNA: 20 CSOC Medium: 4 C or room temperatureNalgene Single-Use PETG 125-mL non-baffled, ventedErlenmeyer Flasks[5][1][2][3][4][5]24Room temperatureSome components are not available in all regions.Store the frozen cells in liquid nitrogen until ready to use. Do not store the cells at 80 C.Product not included in Cat. No. A39111In TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0Product not available in all global regions.ExpiSf Expression System User Guide7
Product informationComponents of ExpiSf Expression SystemComponents of ExpiSf Expression SystemExpiSf ExpressionSystemThe ExpiSf Expression System is designed to allow high-density infection ofsuspension ExpiSf9 cells in a chemically defined, yeastolate-free, serum-free, proteinfree, animal origin-free medium. The system includes ExpiSf9 cells that have beenadapted to serum-free, high-density suspension culture in ExpiSf CD Medium.Transfection, baculovirus production, and recombinant protein expressionexperiments can be performed directly in ExpiSf CD Medium. The ExpiSf Expression System Starter Kit provides cells, culture medium, and reagents to infect1 liter of cell culture. The kit also includes the pFastBac 1 Expression Vector andMAX Efficiency DH10Bac Competent Cells for generation of bacmid DNA usingthe Bac-to-Bac Baculovirus Expression System. Except for the MAX Efficiency DH10Bac Competent Cells, all the components of the ExpiSf Expression System areanimal origin-free.ExpiSf9 cellsThe ExpiSf9 cell line is a derivative of the Gibco Sf9 cell line. ExpiSf9 cells areadapted to high-density suspension culture in ExpiSf CD Medium. Frozen cells aresupplied in, and can be thawed directly into, ExpiSf CD Medium. ExpiSf9 cellsexhibit the following characteristics: Derived from the same parental lineage as the Gibco Sf9 cell line Rapid recovery post-thaw Adapted to high-density, serum-free, suspension growth in the chemicallydefined, yeastolate-free ExpiSf CD Medium Doubling time of approximately 24 hours during log phase growth Maximum cell densities of approximately 20 106 cells/mL in shake flask culture High baculovirus production and protein expression Stable growth and expression over multiple passagesNote: ExpiSf9 cells are also available separately, see Appendix H, “Orderinginformation“.ExpiSf CDMediumExpiSf CD Medium is a chemically defined, yeastolate-free medium developedspecifically for the high-density culture, transfection, and infection of ExpiSf9 cells insuspension.ExpiSf CD Medium exhibits the following features: Formulated as an optimized, c
Vector are conveniently included in the ExpiSf Expression System Starter Kit for expression of your gene of interest in ExpiSf9 cells. pFastBac 1 Expression Vector pFastBac 1 Expression Vector is a non-fusion donor plasmid that is used to clone your gene of interest using restriction enzyme digestion and ligation. Gene expression
Thermo Fisher Scientific Legal Entity Reference Guide Issue Date: April 1, 2014 Expiration Date: October 30, 2014 3 Introduction The Thermo Fisher Scientific Legal Entity Reference Guide is a map to the legal entities within Thermo Fisher. It presents two views of our company information: 1- A cross reference of former brands and product lines
Thermo Fisher Scientific assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. This manual supersedes all previous editions. No liability for consequential damages Thermo Fisher Scientific shall not be liable for any
Preface Safety and Special Notices ii Thermo Scientific BindIt Software for KingFisher Instruments User Manual Thermo Scientific Thermo Scientific KingFisher Presto User Manual (Cat. no. N17413) Thermo Scientific KingFisher 96 User Manual (Cat. no. 15018890) The Thermo Scientific BindIt Software user manual can be found in PDF format in the
12 Thermo Scientific Multiskan GO User Manual Thermo Fisher Scientific The side view of the Multiskan GO with cuvette instrument is shown in Figure 2-4. Figure 2-4. Multiskan GO with cuvette side view The Multiskan GO supports all common 96- and 384-well plates with and without lids as well as low-volume plates (preferably a high base type).
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Thermo Fisher. Guaranteed Success and The Technical Edge are service marks of Thermo Fisher. PerpHecT meters are protected by U.S. patent 6,168,707. PerpHecT ROSS are protected by U.S. patent 6,168,707. ORION Series A meters and 900A printer are protected by U.S. patents 5,198,093, D334,208 and D346,753.
FB 70155 aspirator (Thermo Fisher Scientific, Fisher Scientific, catalog number: 11533485) 13. Grant Instruments water bath (Grant Instruments, model: OLS200) 14. Thermo Scientific Heracell. TM. 150i humidified tissue culture incubator (set at 37 C and 5% CO. 2) (Thermo Fisher
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