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.
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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 : lacZ
gene (Bluo-gal or X-gal) 2. Time-saving expression to identify and
purify a recombinant virus
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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
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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)
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Bacmid -baculovirus shuttle vector AcMNPV bacmid (136kb) Kan
mini-F kanamycin resistance marker LacZ-mini-attTn7 ( a short
segment containing the attachment site for the bacterial transposon
Tn7) a low-copy-number mini- Fertility replicon Donor
plasmid(pFastBac) (6.7kb) SV40 P(A) gusA Gen Ppolh Tn7R Multiple
cloning site SV40 polyadenylation signal Tn7R(bacteria transposon)
gusA Ppolh(promoter) Gentamicin resistance gene
LacZ-mini-attTn7
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site-specific transposition transposed donor vector sequence
Bacterial primer sequence
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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.
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Diagram of the Bac-to-Bac system
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Bac-to-Bac Flow Chart pFastBac donor plasmid Clone gene of
interest pFastBac Recombinant Transform into E.coli DH10Bac E.Coli
Colonies +Rec Bacmid Grow overnight culture Isolate Rec. Bacmid DNA
(do not freeze&thaw) Rec. Baculovirus Transfection into insect
cells
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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.
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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 10 5 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 27C for 5 hours. 5. Remove
the transfection mixture and replace with 2 ml of Insect medium
containing antibiotics. Incubate cells at 27C for 48 hours. 6.
Harvest virus at 48-72 hours post-transfection.
Slide 16 72 hours) Cell lysis, signs of clearing in the
monolayer">
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
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Sf9 control cells Tranfected sf9 cells
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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 +4C, 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.
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Storage information Store viral stock at +4C, 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 -80C for later reamplification. Do not store routinely
used viral stocks at temperatures below +4C. Repeated freeze/thaw
cycles can result in a 10- to 100- fold decrease in virus titer.
Plaque purify your baculoviral construct, if desired
Slide 20
Expression of Recombinant Protein Once you have generated a
baculoviral stock with a suitable titer (e.g. 1 x 10 8 pfu/ml), you
are ready to use the baculoviral stock to infect insect cells and
assay for expression of your recombinant protein.
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Analyzing Protein Expression To detect expression of your
protein by western blot analysis, you may use a Monoclonal antibody
to your protein of interest.
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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.
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Two transfer vectors, each expressing two genes recombine with
the baculovirus genome maintained in E. coli as a bacmid. Two
genetic loci in the baculovirus genome are used, recombination is
controlled by Tn7 transposition the bacmid DNA is transfected into
insect cells, where it directs expression of all four genes at high
levels. The improved expression characteristics of the eukaryotic
expression system promote assembly of correctly processed and
folded tetrameric protein complexes.
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Presented by: Dr. Fotouhi Virologist
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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
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Insect Medium Graces Insect medium- unsupplemented but contains
L- glutamine Graces 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)
Slide 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 0 2 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
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Biological Hood
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Types of cell culturing Monolayer culture Suspension
culture
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Seeding density of cells Disk/flaskSeeding density (cell
number) Vol. of medium (ml) 35mm dia. Petri dish 1x 105 1.5-2 60mm
dia. Petri dish 2 x 105 3-4 25 cm2 flask 1x 106 4-5 75 cm2
flask.5-1 x 107 10 Spinner culture1-2 x 105/ml 40-500
Slide 35
Methods of sub culturing adherent cells Three methods to
dislodge monolayers in adherent cell culture - Pipeting - Tapping
the layer -Trypsinizing
Slide 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
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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
Slide 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
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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
Slide 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
Slide 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
Slide 43
Cryopreseravtion of cells Freezing cells should be 90% viable
and 80- 90%confluent Freezing medium should have 60% Graces insect
medium supplemented with 30%FBS & 10% DMSO
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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
Slide 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
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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
Slide 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