Baculovirus Expression Vector System BEVS

1. Baculovirus Expression Vector System (BEVS)

2. Baculovirus A family of large rod-shaped viruses Circular double-stranded genome ranging from 80-180 kbp. AcMNPV : Autographa califonica multiple nuclear polyhedrosis virus : the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle. : the strong polyhedrin promoter-> the transcriptional control.

3. Bac-to-Bac Baculovirus Expression System An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins Advantage 1. recombinant virus DNA isolated from selected colonies is not mixed with parental, nonrecombinant virus - Easy colony screening : lacZa gene (Bluo-gal or X-gal) 2. Time-saving expression to identify and purify a recombinant virus

4. Bac-to-Bac Baculovirus Expression System Used vectors: 1. Donor plasmid vector into which the gene(s) of interest will be cloned 2. Baculovirus shuttle vector (bacmid) : mini-attTn7 target site, lacZ, kanamycin resistance marker 3. Helper plasmid : transposase, tetracycline resistance marker

5. pFastBac1-Transfer vector, donor plasmid Multiple cloning site SV40 polyadenylation signal Tn7L f1 origin (f1 intergenic region) Ampicillin resistance gene pUC origin Tn7R Gentamicin resistance gene (complementary strand) Polyhedrin promoter (PPH)

7. 7

8. 8

9. Bacmid -baculovirus shuttle vector

10. site-specific transposition

11. Transposon & transpostion Sequences of DNA that can move around to different positions within the genome of a single cell. A process called transposition Transposase: an enzyme Inverted repeats(IR): a sequence of nucleotides that is the reversed complement of another sequence further downstream.

12. Diagram of the Bac-to-Bac system

13. �Bac-to-Bac� Flow Chart pFastBac donor plasmid

14. Analyzing Recombinant Bacmid DNA analyze recombinant bacmid DNA using PCR Note: It is possible to verify successful transposition to the bacmid by using agarose gel electrophoresis to look for the presence of high molecular weight DNA. This method is less reliable than performing PCR analysis as high molecular weight DNA can be difficult to visualize.

15. Transfection  Transfecting Insect Cells with Baculovirus DNA, procedure to transfect Sf9 insect cells in a 6-well format .You will need log-phase cells with >95% viability to perform a successful transfection. 1. Plate 5 x 105 Sf9 cells in 2 ml of Insect medium containing antibiotics. Allow cells to attach for at least 1 hour. 2. For each transfection sample, prepare complexes as follows: a. Dilute 1-2 �g of baculovirus DNA in 100 �l of Insect medium without antibiotics. b. Mix Cellfectin� before use, then dilute 1.5-9 �l in 100 �l of Insect medium without antibiotics. c. Combine the diluted DNA with diluted Cellfectin� (total volume = 200 �l). Mix gently and incubate for 15-45 minutes at room temperature (solution may appear cloudy). 3. Remove the growth medium from the cells and wash once with Insect medium without antibiotics. Remove the wash medium. 4. Add 0.8 ml of Insect medium to the complexes (Step 2c), mix gently and add to the cells. Incubate cells at 27�C for 5 hours. 5. Remove the transfection mixture and replace with 2 ml of Insect medium containing antibiotics. Incubate cells at 27�C for 48 hours. 6. Harvest virus at 48-72 hours post-transfection.

16. Signs of Infection Phenotype Description Increased cell diameter , 25-50% increase in cell diameter Early (first 24 hours) Increased size of cell nuclei may appear to "fill" the cells. Stop of cell growth compared to a cell-only control Late (24-72 hours)Granular appearance, Signs of viral budding; vesicular appearance of cells Detachment Cells release from the plate or flask Very Late (>72 hours) Cell lysis, signs of clearing in the monolayer

17. Sf9 control cells Tranfected sf9 cells

18. Preparing the P1Viral Stock 1. Once the signs of late stage infection (e.g. 72 hours post-transfection) occurred, collect the medium containing virus from each well (~2 ml) and transfer to sterile 15 ml snap-cap tubes.Centrifuge the tubes at 500 x g for 5 minutes to remove cells and large debris. 2. Transfer the clarified supernatant to fresh 15 ml snap-cap tubes. This is the P1 viral stock. Store at +4�C, protected from light. 3. amplify the p1 viral stock and prepare p2 and p3 viral stock(low MOI) Note: If you wish to concentrate your viral stock to obtain a higher titer, you may filter your viral supernatant through a 0.2 �m, low protein binding filter after the low-speed centrifugation step, if desired.

19. Storage information � Store viral stock at +4�C, protected from light. � If medium is serum-free ,add fetal bovine serum to a final concentration of 2%. Serum proteins act as substrates for proteases. � For long-term storage, store an aliquot of the viral stock at -80�C for later reamplification. � Do not store routinely used viral stocks at temperatures below +4�C. Repeated freeze/thaw cycles can result in a 10- to 100-fold decrease in virus titer. � Plaque purify your baculoviral construct, if desired

20. Expression of Recombinant Protein Once you have generated a baculoviral stock with a suitable titer (e.g. 1 x 108 pfu/ml), you are ready to use the baculoviral stock to infect insect cells and assay for expression of your recombinant protein.

21. Analyzing Protein Expression To detect expression of your protein by western blot analysis, you may use a Monoclonal antibody to your protein of interest.

22. Purifying Recombinant Protein You may use any method of choice to purify your recombinant protein of interest. Note: If you have cloned your gene of interest in frame with the 6xHis tag, you may purify your recombinant protein using a metal chelating resin such as ProBond or Ni-NTA .

27. Insect cells Presented by: Dr. Fotouhi Virologist

28. Insects & Insect cells Baculovirus infects lepidopteran insects (butterflies ) and insect cell lines Commonly used cell lines are sf9 & sf21 derived from the pupal ovarian tissue of the fall army worm spodoptera frugiperda and high five derived from the ovarian cells of the cabbage looper

29. Insect Medium Grace�s Insect medium- unsupplemented but contains L-glutamine Grace�s Insect medium supplemented-contains additional TC yeastolate & Lactalbumin hydrolysate Trichoplusia ni Medium formulation hink (TNM-FH) Serum Free Medium(SFM) Aditives: FBS 10-20%, L-Glutamine, non-essential amino acids, antibiotics(penecillin 100 unit/ml, streptomycin 100 ug/ml), antimycotic (fungine 10-50 ugml)

30. Requirements for proper cell culture Temperature- Optimal range is 27-28 C pH- Optimal range is 6.1 to 6.4 Aeration-Requires passive 02 diffusion for optimal growth & recombinant protein expression Osmolality- Optimum is 345-380 mOsm/kg FBS- Working with suspension culture it is advisable to use (10-20% FBS) to gave protection from cellular shear forces

31. Biological Hood

33. Types of cell culturing Monolayer culture Suspension culture

34. Seeding density of cells

35. Methods of sub culturing adherent cells Three methods to dislodge monolayers in adherent cell culture - Pipeting - Tapping the layer -Trypsinizing

36. Procedure of monolayer sub culture Monolayer should reach to confluency in 2-4 days. Aspirate medium & floating cells from a confluent monolayer & discard them. Add 4ml of RT complete growth medium to each 25cm2 flask(12 ml to a 75 cm2 flask) Resuspend cells by pipetting the medium across the monolayer with a Pasteur pipette. (Enzymatic dissociation is not recommended) Observe cell monolayer using an inverted microscope to ensure adequate cell detachment

37. Contd.. Perform viable cells count on harvested cells. Inoculate cells at 2 x 105 viable cells/ml into respective culture vessels. Inoculate cultures kept at 25-28 C On day 4 post-planting, aspirate the spent medium from one side of the monolayer & subculture the flask With slower growing cell lines, it may be necessary to feed the flasks on day 3-4 post planting Subculture the flasks when the monolayer reaches 80-100% confluency, approx 2-3 days post planting

38. Working with suspension culture Insect cells are not generally anchorage dependent & can be well adapted to suspension culture Prior to establish a spinner culture, cells are maintained firstly as healthy adherent cells. Use a spinner flask with a vertical impeller Culture volume should not exceed half of the volume of the flask Use of surfactant to decrease shearing e.g. Pluronic F-68

40. Contd.. Not necessary to change medium regularly. Sub culturing requires the removal of cell suspension & the addition of medium Impeller should be rotating regularly Impeller should be submerged 1 cm or more to ensure adequate aeration Cell viability of 95% is required Minimum density of 1 x 106 cells/ml is required

41. Contd� Keep record of the passage number. After 30 passage or more (2-3 months), cells doubling time increased and also loose their viability and infectivity. Keep a cell log, to do so one should have a knowledge of following; date of initiation of culture, lot number date of passage & passage number density & viability at passage comment on cell appearance medium & its lot number

42. Initiation of culture with freezed cells Thaw the frozen suspension rapidly in a water bath at 28 C Seed the cells into a culture flask (1 x 106) containing medium 5 ml TC 100 medium Incubate at 28 C for 5 hrs Change with fresh medium Incubate again, until it reach confluence Subculture it for experimental purpose

43. Cryopreseravtion of cells Freezing cells should be 90% viable and 80-90%confluent Freezing medium should have 60% Grace�s insect medium supplemented with 30%FBS & 10% DMSO

44. Procedure Count cells using haemocytometer Placed cryovials on ice & label them Centrifuge cells at 400-600 g for 10 mts at RT. Remove the supernatant Resuspend the cells to the given density in the freezing medium Transfer 1 ml of the cell suspension to sterile cryovials Place at -20 C for 1 hr then transfer to -80 C for 24-48 hrs & then finally store at Liquid nitrogen

45. Do and Don'ts Check cells daily until a confluent monolayer is formed. Passage cells at confluency only, as cells will be easy to dislodge & shows better viability Do not overgrow cells, it results in decreased viability Do not splits cells too for. Densities lower than 20% confluency inhibit growth Passage the cells only in log phase, log phase growth can be maintained by splitting cells in 1:5 dilution

46. Basic aseptic conditions If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger Work either left to right or vice versa, so that all material goes to one side, once finished Clean up spills immediately & always leave the work place neat & tidy

47. Contd.. Possibly keep cultures free of antibiotics in order to be able to recognize the contamination Never use the same media bottle for different Insect cell lines. If caps are dropped or bottles unconditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C Switch on the laminar flow cabinet 20 mts prior to start working Cell cultures which are frequently used should be subcultered & stored as duplicate strains