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Functional Genomics
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
TABLE OF CONTENTS
TABLE OF CONTENTS..........................................................................................3
ACKNOWLEDGMENTS .....................................................................................5
PARTICIPANTS ...................................................................................................6
VECTOR CONSTRUCTION..............................................................................29
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REFERENCES ....................................................................................................49
APPENDIX .........................................................................................................51
Vector Maps...................................................................................................................................................51
Reagents .........................................................................................................................................................53
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ACKNOWLEDGMENTS
We are grateful to the National Center for Genetic Engineering and Biotechnology
(BIOTEC) for hosting this workshop on their Bangkok campus. This workshop was
made possible by the generous funding of Howard Hughes Medical Institute
(HHMI), BioMalPar and WHO Special Programme for Research and Training in
Tropical Diseases (TDR), and we are especially grateful to Jill Conley (HHMI),
Artur Scherf (BioMalPar) and Ayo Oduola (TDR) for their enthusiastic support.
We thank the Walter and Eliza Hall Institute of Medical Research (WEHI) and
BIOTEC for in-kind support and special thanks to Sumalee Kamchonwongpaisan,
Chairat Uthaipibull and Marian Cravino for their tremendous assistance in
organization of the workshop. We also thank other members of our laboratories
for compiling this manual and supplying vectors and ideas, especially Alex Maier,
Julie Healer, Chris Tonkin, Paul Gilson, Tania de Koning Ward, Matt O’Neill,
Monica Brown, Joanne McCoubrie, Tony Triglia, Melanie Rug and Rebecca
O’Donnell.
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PARTICIPANTS
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COURSE TIMETABLE
18-Mar 19-Mar 20-Mar 21-Mar 22-Mar 23-Mar 24-Mar 25-Mar 26-Mar
Sunday Monday Tuesday Wednesday Thursday Friday Saturday Sunday Monday
9:00 TL2 -Vectors & 9:00 Transfection of P. 9:00 TL4 - Promoters 9:00 TL5 - Southern Blot 9:00 TL7 - Inducible FREE 9:00 TL8 - New vectors 9:00 Southern detection,
Cloning Methods berghei (BC) Workshop (CT) expression (PG) & future possibilities Smear Pf
(CT) (BC /AW) transgenic cultures,
examine GFP
Feed P.falciparum 10:00 gDNA digests (Split 10:00 Pour & load gels and 10:00 Smear Transfectant inducibles
cultures into two groups for run, smear Pf cultures
two labs) transfectants and
inducible system +ATC
(microscope)
10:00 COFFEE 10:30 COFFEE 10:00 COFFEE 10:50 COFFEE 10:50 COFFEE 10:50 COFFEE 10:30 COFFEE
10:20 SPLIT INTO TWO 10:50 Transfection of P. 10:20 P. berghei - drug 11:10 SPLIT INTO TWO 11:10 SETUP INVASION 11:10 Hybridize Southern 10:50 Smear cultures,
GROUPS: berghei, feed Pf selection, smear GROUPS: Smear & ASSAY: Count IA blot with probe examine inducibles
A: Stable cultures transfectants and feed transfectants, lines, adjust GFP, Southern
transfection of Pf feed, examine GFP examine GFP parasitemia detection
B: Intro to in Pf
PlasmoDB and
VectorNTI
11:10 Swap over groups 12:00 SS - BMP - 12:00 SS - BMP - Oliver 12:00 SS - Dave Fidock 12:00 SS - Manoj
Christian Doerig Billker Duraisingh
13:00 LUNCH 13:00 LUNCH 13:00 LUNCH 13:00 LUNCH 13:00 LUNCH 13:00 LUNCH 13:00 LUNCH
14:00 Welcome & 13:30 Set O/N culture of 13:30 SS - BMP Elena 13:30 SS-BMP - Robert 13:30 SS - BMP - 13:30 SPLIT INTO TWO 13:30 ANALYSE INVASION 13:30 Analyse transient
Introduction P. berghei (two Levashina Menard Dominique Soldati GROUPS: ASSAY: transfections and
(BC) demonstrations)
14:45 Short student 14:50 TL2 - Derivation 14:30 SS - BMP - Kai 14:30 Finish off 14:30 SPLIT INTO TWO A: Setup invasion Smear Invasion Analyse Pb
introductions of stable Pf Lines Matushewski experiments from GROUPS. A: Transient assay (enzyme assay and count transfectants and
(BC) the morning transfection of treatments) smear & discard Pf
(Computer time) luciferase plasmid in B: Blot southern cultures
gels
Pf.
15:00 TL1 -Overview 15:30 Chalk talk with B: Southern blot
(AC) BMP speakers mapping on
computer
16:00 COFFEE 15:50 COFFEE 15:50 COFFEE 15:50 COFFEE 16:20 COFFEE 15:50 COFFEE 15:50 COFFEE 15:50 COFFEE
16:20 Preparation of 16:10 Feed P. falciparum 16:10 Own construct 16:10 Image software 16:40 Swap over groups - 16:10 Swap over groups 16:10 SPLIT INTO TWO 16:10 Final Discussion
parasites and DNA transfection design, ImageJ example and take out gDNA GROUPS:
for transfection dishes, add drug construct design digests A: Harvest parasites
and freeze from
17:00 TL3 - Derivation transients
of stable Pb lines B: Computer exercise
(AW/TdKW)
DEPART
18:30 DINNER/ 18:30 18:30 18:30 CULTURAL 18:30 18:30 18:30 18:30
DINNER DINNER DINNER DINNER/CRUISE DINNER DINNER
RECEPTION EVENING
AC - Alan Cowman AW - Andy Waters BC - Brendan Crabb CT - Chris Tonkin JH - Julie Healer PG - Paul Gilson TdkW - Tania de Koning-Ward
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Plasmodium falciparum
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TRANSFECTION
2. Allow the pellet to dry for 5 min in a laminar flow hood. Resuspend DNA in
15-30 µl of sterile TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA). It is essential
that the DNA be fully dissolved in the buffer before adding further
solutions.
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3. Fresh media and the appropriate drug are added to cultures daily for the
first 4 days then every 2 days until parasite establishment.
4. Smear parasite culture on day 2 post transfection to check for the presence
of rings (there should be almost no rings present).
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VECTORS
3. Grow the culture until parasites re-appear and until parasite growth is
firmly established.
4. Analyse chromosomes and genomic DNA of the parasites using PFGE and
Southern blotting to determine if integration into the appropriate gene has
occurred in these parasites.
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(1) PCR. This approach can be used for all requirements. However, for a number
of reasons we believe that its use is limited and that the technique should be used
as a guide only and results have to be confirmed using other techniques. For
example, detection of the transfection plasmid by PCR using oligonucleotides
specific for a unique sequence (such as a targeting sequence) is confounded by the
presence of residual DNA left over from the original transfection. This DNA can be
destroyed by pre-digestion of the gDNA with Dpn I, a restriction enzyme with a
frequently found recognition sequence that cleaves only methylated (such as that
replicated in E.coli) and not un-methylated (parasite replicated) DNA, although
DpnI digestion is unlikely to be 100% efficient. PCR is particularly useful to detect
the presence of homologous integration events using a combination of a plasmid-
specific oligonucleotide (not specific to the gene targeting sequence) and one
directed to genomic sequence located immediately outside of the gene targeting
fragment found in the plasmid. The presence of such a product (which should be
sequenced for confirmation) demonstrates that homologous integration has
indeed occurred. Using this approach, however, it is not possible to determine the
proportion of the parasites that possess integrated forms of the plasmid.
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Therefore hybridisation of duplicate Southern blots of a pulsed field gel with the
plasmid backbone and the positive selectable marker sequence will differentiate
between integration by single and double crossover recombination. Only the
positive selectable marker will hybridise to double crossover integration events
and the plasmid backbone will not.
It can take several months to generate a gene knockout parasite so before any
phenotype analysis is carried out it can be worthwhile to confirm that the parent
and the knockout have an identical karyotype. Karyotype analysis can be done by
ethidium bromide staining chromosomes that have been resolved by PFGE. This
will also detect any chromosomal rearrangements that have occurred during the
transfection experiment. Further comparison of the genome of the parent and
knockout parasites can be done by hybridisation of the repetitive probe rep20 to
genomic DNA digested with HindIII.
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(NB: For the best yield of genomic DNA use a culture with 6-10% trophozoites.
The volumes in brackets are for a 10 ml culture)
1. Spin culture at 1200 rpm for 5 minutes to pellet parasitised red blood cells
and remove supernatant.
3. Add 1 volume of 18% SDS (0.4 mls) and mix thoroughly then let sit for 2-3
minutes.
6. Remove aqueous phase into a clean corex tube and ethanol precipitate by
adding 1/10 volume of 3M Na Acetate pH5.0 (250 µl) and 2.5 volumes of
ethanol (6.5 ml).
7. Leave at –20oC for at least 1 hour, but the preparation could be stored
overnight at –20oC at this stage.
12. Extract once with chloroform by adding 600 µl of chloroform, mixing and
spinning at 4,000 rpm for 3 minutes then removing aqueous phase to a
clean tube.
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14. Leave at –20oC for at least 1 hr, but the preparation could be stored
overnight at –20oC at this stage.
16. Remove supernatant and wash DNA pellet with 1 ml of 70% ethanol
Alternatively the DNeasy tissue kit (Qiagen) could be used – most of the solutions
are provided by the kit. A 10ml culture >5% mature parasites will yield around 10-
20µg DNA.
1. Saponin lyse the culture and wash the pellet once in PBS, pH7.4.
5. Vortex for 15sec. Add 400µl buffer AL to the sample and mix vigorously by
vortexing.
6. Pipet the mixture into the DNeasy Mini Spin Column placed in a 2ml
collection tube. Centrifuge at 8000rpm (=6000 x g) for 1min. Discard flow-
through and collection tube.
7. Place the DNeasy Mini Spin Column in a new 2ml collection tube, add
500µl buffer AW1, and centrifuge for 1min at 8000rpm (=6000 x g).
Discard flow-through and collection tube.
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8. Place the DNeasy Mini Spin Column in a new 2ml collection tube, add
500µl of buffer AW2, and centrifuge for 3min at 15000rpm (=20000 x g) to
dry the DNeasy membrane. Discard flow-through and collection tube.
9. Place the DNeasy Mini spin column in a clean 1.5ml microcentrifuge tube
and pipet 150µl buffer AE directly onto the DNeasy membrane. Incubate at
RT for 1min and then centrifuge for 1min at 8000rpm (=6000 x g) to elute.
Before setting up the DIG PCR labelling reaction make a 1:3 dilution of the PCR
DIG labelling mix (vial 2) with the dNTP stock solution (vial 4). This gives a 1:9
working solution since the DIG PCR labelling mix stock solution (vial 2) is a
dilution of 1:3 to begin with.
NB. If you don’t get any PCR product with the 1:3 dilution of the DIG PCR
labelling mix try a dilution of 1:6 of the DIG PCR labelling mix (vial 2) i.e a 1:18
dilute working solution. Do not use dilutions of the PCR DIG labelling mix below
1:20.
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Mix reagents and centrifuge briefly to collect sample at bottom of tube. Place
samples in thermal cycler and start PCR:
1 95oC 5min
2 95oC 30s
3 60oC 30s
4 62oC 2.5 - 4 mins
5 Return to 2 34x
6 72oC 7min
7 4o C hold
3. Run 5µl of PCR reaction on 1% agarose gel (Kit Control will run at 500-
550bp). Size of DIG labelled probe will be larger than unlabelled probe and
will stain a little bit weaker.
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1. Run digested genomic DNA (1.0-5.0 µg) on a 0.8% agarose gel o/n at 17V.
3. Depurinate DNA in 0.125M HCl (5ml conc. HCl/400ml DDW) for 20min.
7. Build up blot, pre-wet Hybond-N and 1st sheet in DDW (evenly), Blot O/N
(or at least 4h).
11. Rinse membrane briefly in DDW and allow to air dry (membrane can be
stored dry at 4oC).
Pre-hybridization: (do not allow the blot to dry out once you start)
Thyb=Tm-(20 to 25°C)
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14. Place correct amount of DIG Easy Hyb in tube and place tube in water-bath
set at hyb temperature.
Hybridisation
16. Prepare hyb solution: add appropriate amount of labelled probe (0.5-1µl)
per ml final hyb solution) + 50µl H2O in Eppendorf tube, heat to 95°C for
5min, cool quickly in an ice-bath
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18. Pour out pre-hybridisation buffer, add immediately hyb solution containing
probe to bag, remove air-bubbles and seal bag.
20. Incubate bag O/N at appropriate hyb temp, agitate blot gently.
21. Pour hybridisation solution off (store in Falcon tube at -20°C/can be reused
3-5 times).
22. Wash membrane twice with 2 x SSC at RT for 5 min in a shaking container
(make sure membrane does not dry out).
26. Wash membrane in 100ml washing buffer (0.1M maleic acid, 0.15M NaCl
buffer, pH7.5, 0.3% Tween-20) at RT for 15 min.
27. Wash membrane in 100ml washing buffer (0.1M maleic acid, 0.15M NaCl
buffer, pH7.5, 0.3% Tween-20) at RT for 15 min.
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29. Remove excess liquid from membrane by dripping one corner dry onto a
tissue, transfer into hybridisation bag, add 2ml CSPD onto membrane,
remove air-bubbles by stroking it with a wet tissue, cover immediately.
31. Squeeze out excess liquid, remove air-bubbles, seal bag and put in exposure
cassette and place at 37°C for 10 min.
Reusing a probe
1. Heat and mix the DIG Easy Hyb solution/DIG probe at 64°C for 10
minutes.
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BLOCKS
Chromosome blocks are agarose plugs that contain chromosomal DNA molecules
that can be resolved by pulsed field electrophoresis. Standard procedures for DNA
preparation do not yield chromosome-sized DNA molecules because high
molecular weight DNA is sheared by mechanical forces during preparation.
P.falciparum chromosome blocks are prepared by embedding parasites in agarose
followed by in situ lysis and deproteinisation.
1. Pellet parasitised red blood cells 1200 rpm 5 minutes and discard
supernatant.
2. Saponin lyse the red blood cells in 1.5 volumes (600 µl) of 0.15%saponin in
RPMI-Hepes on ice for 5 minutes.
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7. Pipette mixture into block cast and allow the agarose blocks to set on ice.
8. Push the blocks into lysis buffer 0.5M EDTA, 10mM Tris pH 8.0, 1%
sarkosyl, 2 mg/ml proteinase K (proteinase K added fresh just prior to use).
Allow approximately 1 ml of lysis buffer for up to 500 µl of blocks.
The following protocol is suitable for a BioRad CHEF pulsed field electrophoresis
apparatus. Gels are run at 13°C.
3. Prepare 100 mls of 1% agarose in 0.5X TBE* (make the volume up to 100 mls
with H2O after boiling in a microwave oven to melt the agarose).
4. Allow the agarose solution to cool to approximately 60oC then pour into the
casting stand.
5. Allow the gel to set at room temperature then carefully remove comb.
7. Load the samples into wells and seal with 1% LMA agarose in 0.5X TBE*
(which has been melted and cooled to approximately 50°C).
8. Pour the remainder of the 0.5X TBE* buffer into the tank and turn on cooling
system.
10. Set the appropriate running conditions (see below) and run the gel.
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11. To visualise the chromosomes, remove the gel into a suitable container and
stain in a 0.5 – 1 µg/ml ethidium bromide solution in H2O or 0.5X TBE for at
least 30 mins.
13. DNA fragments larger than 20kb must be cleaved for efficient transfer to
hybridisation membranes. Prior to transfer, DNA fragments separated by
pulsed field electrophoresis are nicked by acid treatment or UV irradiation (5
minutes on a short wavelength UV transilluminator).
14. The DNA can then be transferred and hybridised using standard procedures.
1% agarose in 0.5 x TBE, 60 – 120 s pulse, 6 V/cm (200 volts), 24 hour run.
Episomes generally migrate around 35 mm from the wells. These conditions can
be useful for detecting the presence or absence of episomes and integration events
into loci on chromosomes 1 and 2.
1% agarose in 0.5 x TBE, 225 sec. pulse, 4.2 V/cm (140 volts), 60 hour run.
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1% agarose in 1X TAE, 360 – 800 s pulse, 3 V/cm (100 volts), 96 hour run.
Episomes generally run off these gels so these conditions are only useful for
detecting integration events into loci on chromosomes 11 –14.
Prior to embarking on the transfection experiments, you will have already given
some thought as to what possible phenotypic changes may result from the genetic
manipulation you will make to the parasites. Obviously, phenotypic analyses will
depend on which gene you are studying. Below is a method we use to assess
whether knockout of putative invasion-related genes results in an altered
erythrocyte receptor use [10],[8], [11], [12], [13].
It is critical that parasite lines are tightly synchronised and growing well before an
invasion assay is set up. You will be given two P. falciparum parasite lines, one
parental type W2mef and the other W2mefΔ175, in which the gene for EBA175 has
been deleted by double cross-over recombination using the pHTK vector [10].
These will be tested for invasion into 4 different erythrocyte populations:
untreated, neuraminidase-treated, chymotrypsin-treated and trypsin-treated. The
protease enzyme treatments remove different classes of erythrocyte surface
proteins, whereas neuraminidase removes specific sialic acid glycans from surface
proteins such as the glycophorins. It is advisable to treat parasitised cells at the
early ring stage, since these suffer less adversely from the various treatments and
washes.
Day 1
1. Smear cultures and synchronise using 5% sorbitol 2 days before the assay
set up. Adjust parasitaemia to 1%.
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Day 3
2. On the morning of the assay set up, synchronise parasite stocks with
sorbitol and smear cultures. Adjust parasitaemia to 1% and haematocrit to
4% with fresh erythrocytes. From this point on, it is important to take care
when removing supernatants, as we want to maintain the same
parasitaemia and haematocrit across different parasite lines and
subsequent treatments.
5. Incubate 1 hour at 37°C with gentle shaking. While cells are incubating,
label 10ml tubes with parasite line and treatment. After 1 hour, resuspend
trypsin and chymotrypsin-treated cells in 100 μl inhibitor solution.
Incubate at 37°C for 10 mins. Resuspend all cells and transfer to labelled
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10ml tubes for washing steps. Add 5ml wash buffer (WASH 1). Spin down
and remove medium (WASH 2). Resuspend again in 5ml medium, spin and
remove medium (WASH 3). Resuspend to exactly 2.5ml COMPLETE
medium.
6. Label a sterile 96-well U-bottom plate, leaving outside wells free, these will
be filled with medium to prevent drying out of assay. Set up each parasite
line in columns, with triplicate wells for each treatment (4 rows). There will
be a total of 12 wells for each parasite line, each with 4 treatments: no
enzyme, neuraminidase, chymotrypsin and trypsin. In addition set up a
couple of control wells with untreated W2mef parasites. Place plates back in
incubator until day 5.
Day 4
Free day.
Day 5
7. Smear control wells, if parasites have reinvaded (rings), smear all wells,
giemsa stain slides and count invasion events. Percentage invasion of each
parasite line into each population of rbc should be calculated by counting
invasion events per 1000 erythrocytes. Compare parental and mutant
parasite lines for a switch in receptor usage.
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VECTOR CONSTRUCTION
1. pHH1. The pHH1 vector [16], its parent vector pHC1 and derivatives [17, 18]
have been useful for gene targeting and for transgene expression to analyse
protein trafficking, merozoite invasion and drug resistance. This vector
allows integration of the plasmid into the genome of P.falciparum by single-
crossover recombination. Although very useful, this strategy has a major
drawback in that it does not allow selection of gene disruptions that are not
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3. pCC vectors. pCC vectors allow - like the pTK vector - the integration via
double crossover recombination [20]. Instead of the thymidine kinase gene
they use cytosine deaminase gene as a negative selectable marker. This
selection is more stringent allowing a faster and more reliable selection
procedure. In addition the pCC vector series is modular making the exchange
of the positive selectable marker cassettes easier (see below) and therefore
are used for subsequent ‘knockouts’ of different genes in the same parasite
line.
4. pHH2. The use of GFP tagged proteins has been an important application to
follow the trafficking pathway of proteins in live P.falciparum-infected
erythrocytes [18, 21]. The transfection vector pHH2 allows cloning of
sequences into a gene cassette to obtain expression of proteins to GFP. This
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vector uses the promoter from the hsp86 gene, which allows a broad
expression of the GFP in Plasmodium blood stages.
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The large size and high AT content of Plasmodium falciparum transfection vectors
makes cloning very challenging. To overcome these difficulties we have utilised
Invitrogen’s Multisite Gateway™ technology to create a novel set of vectors. These
vectors allow easy assembly of fluorescent protein constructs while allowing
flexibility in promoter strength and timing, and permitting the use of different
fluorescent proteins (and other tags) in conjunction with different targeting
elements (see figure below) [23].
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human DHFR with rep20 in a head-to-tail (-) orientation with the attR3/R4
cassette) and pHBlR-3/4 (HSP86 5’ driving blasticidin-S deaminase in a head to
tail orientation (-) with the attR3/R4 cassette).
At present we have made promoter-ENTR clones that contain the HSP86 5’, CRT
5’ and AMA1 5’. We also have gene tag-ENTR clones containing GFP, eYFP,
monomeric DsRED and 3XHA. However it is easy to create other ENTR clones
containing a favourite promoter or gene tag. We have used these vectors
extensively to make single and double transgenic parasites and they are now
routinely used in many labs.
Protocol Overview
2. Perform PCR reaction, clean-up reaction and mix product with pENTR-
D/TOPO vector.
3. Screen colonies and sequence pENTR-D vector to confirm that your gene
(or part thereof) has no mutations and is otherwise correct.
4. Choose your promoter, gene tag –ENTR clones and your destination clone
containing desired selectable marker.
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dH2O Xμl
--------
Total 20μl
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3. Add 1.5x pellet volume of saponin and place on ice for 10 min.
LUCIFERASE ASSAY
10. Prepare desired amount of Promega’s Stop and Glo Reagent (Renilla
substrate). 100 µl is required for each sample.
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12. Once prepared these substrates must be kept at room temperature and
protected from light.
13. For each sample 20 µl of parasite lysate is aliquotted into assay tubes (for
tube luminometers) or 96 well plates (for plate luminometers). Follow
manufacturer’s instructions for operating protocols.
15. Add 100 µl of Stop and Glo and read luminescence for 24-45s. Addition of
the Renilla reagent quenches the firefly luminescence and simultaneously
activates R. reniformis activity.
VECTOR MAPS
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In this course we will induceably express two proteins tagged with GFP from the
constructs pTGFP-GPI and pTGFPM19 (Figure 3). The pTGFP-GPI fusion protein
contains an endoplasmic reticulum (ER) signal sequence followed by GFP and is
terminated with a signal directing the attachment of a
glycosylphosphatidylinositol (GPI) anchor (Figure 3A). This protein should
traffick to the surface of the P. falciparum schizonts/merozoites and remain
attached the outside of the plasma membrane. The second fusion pTGFPM19, is
the same as the first except that C-terminal fragment of merozite protein 1 (MSP-
119) has been inserted in frame, in between the GFP and the GPI-attachment signal
(Figure 3B).
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Day1
2. To turn expression of the GFP fusion protein on, the ATc must be removed.
This is done by first harvesting the parasite culture and transferring it to a
10 ml tube. The red blood cells infected with ring-stage parasites (iRBCs)
are then pelleted by spinning the culture at 1500g for 5 mins. To
simultaneously synchronize the parasites and remove the ATc the culture
media is removed and replaced with 5 pellet volumes of 5% sorbitol (5%
sorbitol in water). The iRBCs are resuspended and incubated for 5 mins @
37°C. The iRBCs are then pelleted as above and rinsed in 5 volumes of
culture media. Finally the iRBCs are resuspended in 10 mls of media and
1.25 µl of 20µM WR99210 is added before the culture is returned to a 10 ml
Petri dish.
Days 2 and 3
3. It takes some time for the ATc to diffuse out of the RBCs and for its
concentration to drop sufficiently so that the parasites can express the GFP
fusion protein. For this reason it takes 3 days for the expression of the GFP
fusion protein to reach a maximum. To facilitate maximum expression it is
important to change the parasite’s media each day.
Day 4
4. When the parasites have become schizonts for a second time since having
the ATc removed (approx. 3 days later) it is time to look at them under the
microscope. Remove 1 ml of iRBCs resuspended in their culture media and
add 2-5 µl DAPI (50µg/ml) to stain the parasite nuclei.
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5. Spin parasites at 1500g for 5 mins and remove 400 µl media leaving 100 µl
behind in which the iRBCs should be resuspended. Drop 5 µl of iRBCs onto
a microscope slide and gently place a 22 x 64 mm coverslip on top of the
cells. Allow the cells to spread out and place under the microscope.
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Plasmodium berghei
In this section, the methods that are typically used to stably transfect P. berghei
are described. Details have been extracted from the following website
(www.lumc.nl/1040/research/malaria/model.html) and more comprehensive
information can be found on this site.
Since P. berghei cannot be readily cultured in vitro for more than one cycle, rats or
mice infected with P. berghei are used as a source of bloodstage parasites for the
culture and purification of mature schizonts. Introduction of DNA into these
mature stages of P. berghei has so far proven to be more successful than into ring
and trophozoite stages. The most widely used DNA constructs contain the
pyrimethamine resistant form of the T. gondii DHFR/TS gene as a selectable
marker. This enables transfected parasites, which are injected back into mice, to
be selected by treating mice with pyrimethamine. Depending on the desired
outcome, the plasmid DNA is either transfected as undigested circular DNA (for
episomal replication within the parasite) or as linearised DNA. In the latter
scenario, the DNA construct can either be linearised at a unique site located within
the target sequence if attempting to integrate plasmid DNA into the parasite
genome via a single crossover event, or alternatively, the DNA is digested at the
ends of the 5’ and 3’ target sequence (and preferably also within the vector
backbone) so as to remove the plasmid backbone from the rest of the construct in
order to drive a double crossover event.
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
1. For rats, inject around 1.5 × 107 parasites intraperitoneally (i.p) per rat and
these will be ready for harvest around 4 days later (day 4). The parasitemia
at harvest should be between 1-4% (no more). 1 rat (approx 5-8 ml
heartblood) will be sufficient for up to 12 transfections.
2. For mice, inject around 2.5 × 106 parasites i.p into 7-8 new donor mice (or
sufficient mice so as to get around 5 ml of heartblood). Parasitemias should
be between 1-4 % on day 3. This amount of heartblood will be sufficient for
12 transfections.
1. When the parasitemias of the animals reach between 1-4 % heart bleed
animals in the afternoon, using a 23 -G needle with attached syringe
containing 0.1 ml heparin stock solution. Pool blood from all mice/rat into a
50 ml tube containing 5-10 ml complete culture medium.
4. Gas flasks and incubate at 37°C overnight. Flasks can either be continuously
gassed using an ‘automatic’ continuous gassing system whereby the cultures
are continuously gassed throughout the complete culture period using 5%
CO2, 5% O2, 90% N2. Alternatively cultures can be maintained in closed
plastic 500 ml culture flasks that have been gassed once for 2 minutes at the
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
5. (Day 4) At around 9.00 am the following day smear parasites by taking out
300 μl of gently resuspended parasites, spinning briefly in 1.5 ml
microcentrifuge tube, removing supernatant and resuspending parasites in
residual medium. Make smear and stain. Check that the schizonts are nice
and healthy.
1. Split parasite cultures into 50 ml tubes so that have 30-35 ml culture in each.
Prepare 55% Nycodenz solution and gently layer 10 ml of this very carefully
underneath the suspension. For a culture suspension of 150 ml a total
volume of 50 ml of 55% Nycodenz is used (=27.5 ml nycodenz stock solution,
22.5 ml PBS)
2. Spin 20-30 min at 1200 rpm in a swing out rotor at room temp, NO BRAKE
3. Carefully collect the brown layer containing schizonts (and gametocytes and
old trophozoites if present) at the interface with pasteur pipette into new
50ml tube. Uninfected red blood cells will pellet at the bottom of the tube. In
general a total volume of about 30-40 ml is collected. Add around 20 ml of
culture medium from the top of this nycodenz density gradient to help wash
away the nycodenz.
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
2. Allow the pellet to dry and resuspend each DNA construct in 5-10 μl water or
TE buffer (10 mM Tris, 1mM EDTA, pH 8).
1. Add 100 μl of the ‘Human T cell NucleofectorTM solution from the Amaxa kit
to the resuspended DNA.
1. To select for parasites harbouring transfected DNA provide the animals with
drinking water containing pyrimethamine 24-30 hrs after injection of
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
transfected parasites and treat for 4-7 days. Smear the mice every 2nd day
from day 5-6 onwards. The parasitemia the day after transfection usually
ranges between 0.05-3%. After the first 2 drug treatments a rapid drop in
parasitemias occurs to undetectable levels indicating that most of the
parasites do not contain the DNA constructs. In successful experiments using
the Amaxa electroporator the parasitemia increases to levels of 0.1-5%
between days 4 and 7 after transfection. In unsuccessful experiments
parasites are often detected between day 13 –15 after the injection of
transfected parasites. These parasites are usually non-resistant wildtype
parasites that survived the drug treatment protocol.
1. Collect heart blood from 1 mouse with parasitemia around 1-5% using 23-G
needle containing around 0.05-0.1 ml heparin solution in the syringe.
2. Gently mix blood with an equal volume of sterile freezing solution (30%
glycerol made up in PBS).
3. Aliquot between 300-500 μl per cryovial. Leave cryovial at 4°C for 5 min then
store in liquid nitrogen.
1. Heart bleed mice displaying parasitemia of between 5-15% using 23-G needle
containing around 0.05-0.1 ml heparin solution in the syringe and suspend
the blood in 5 ml PBS.
2. Remove leukocytes from the blood by passing the blood suspension through
a Plasmodipur filter (Euro-Diagnostica, www.eurodiagnostica.com) or
through CF11 powder. For removal of leukocytes through a CF11 column:
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
H E B B E A E
The transfection plasmid PbGFPCON is used for the stable expression of GFP. This
construct contains the pyrimethamine-resistant T. gondii DHFR-TS gene for
selection of transgenic parasites and an incomplete copy of the D-SSU-rRNA as a
target region for integration. The vector can be linearised at the unique ApaI site
for integration. The GFP gene is flanked by the EF-1αa promoter and the 3' UTR of
P. berghei DHFR-TS. B: BamHI, E: EcoRI and H: HindIII. Extracted from [28].
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
REFERENCES
[1] Wu Y, Sifri CD, Lei H-H, Su X-S, Wellems TE. Transfection of Plasmodium
falciparum within human red blood cells. Proc Natl Acad Sci USA 1995;92:973-7.
[2] Crabb BS, Cowman AF. Characterization of promoters and stable transfection by
homologous and nonhomologous recombination in Plasmodium falciparum. Proc
Natl Acad Sci USA 1996;93:7289-94.
[3] Wu Y, Kirkman LA, Wellems TE. Transformation of Plasmodium falciparum
malaria parasites by homologous integration of plasmids that confer resistance to
pyrimethamine. Proc Natl Acad Sci USA 1996;93:1130-4.
[4] Fidock DA, Wellems TE. Transformation with human dihydrofolate reductase
renders malaria parasites insensitive to WR99210 but does not affect the intrinsic
activity of proguanil. Proc Natl Acad Sci USA 1997;94:10931-6.
[5] Deitsch K, Driskill C, Wellems T. Transformation of malaria parasites by the
spontaneous uptake and expression of DNA from human erythrocytes. Nucleic
Acids Res 2001;29:850-3.
[6] Baldi DL, Andrews KT, Waller RS, et al. RAP1 controls rhoptry targeting of RAP2
in the malaria parasite Plasmodium falciparum. EMBO J 2000;19:2435-43.
[7] Crabb BS, Cooke BM, Reeder JC, et al. Targeted gene disruption shows that knobs
enable malaria-infected red cells to cytoadhere under physiological shear stress.
Cell 1997;89:287-96.
[8] Gilberger TW, Thompson JK, Triglia T, et al. A novel erythrocyte binding antigen-
175 paralogue from Plasmodium falciparum defines a new trypsin-resistant
receptor on human erythrocytes. J Biol Chem 2003;278:14480-6.
[9] Maier AG, Duraisingh MT, Reeder JC, et al. Plasmodium falciparum erythrocyte
invasion through glycophorin C and selection for Gerbich negativity in human
populations. Nat Med 2003;9:87-92.
[10] Duraisingh MT, Maier AG, Triglia T, Cowman AF. Erythrocyte-binding antigen 175
mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -
independent pathways. Proc Natl Acad Sci U S A 2003;100:4796-801.
[11] Baum J, Maier AG, Good RT, Simpson KM, Cowman AF. Invasion by P.
falciparum merozoites suggests a hierarchy of molecular interactions. PLoS
Pathog 2005;1:e37.
[12] Triglia T, Duraisingh MT, Good RT, Cowman AF. Reticulocyte-binding protein
homologue 1 is required for sialic acid-dependent invasion into human
erythrocytes by Plasmodium falciparum. Mol Microbiol 2005;55:162-74.
[13] Stubbs J, Simpson KM, Triglia T, et al. Molecular mechanism for switching of P.
falciparum invasion pathways into human erythrocytes. Science 2005;309:1384-7.
[14] Ben Mamoun C, Gluzman IY, Goyard S, Beverley SM, Goldberg DE. A set of
independent selectable markers for transfection of the human malaria parasite
Plasmodium falciparum. Proc Natl Acad Sci USA 1999;96:8716-20.
[15] de Koning-Ward TF, Waters AP, Crabb BS. Puromycin-N-acetyltransferase as a
selectable marker for use in Plasmodium falciparum. Mol Biochem Parasitol
2001;117:155-60.
[16] Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh1 modulates sensitivity
and resistance to multiple antimalarials in Plasmodium falciparum. Nature
2000;403:906-9.
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[17] Crabb BS, Triglia T, Waterkeyn JG, Cowman AF. Stable transgene expression in
Plasmodium falciparum. Mol Biochem Parasitol 1997;90:131-44.
[18] Waller RF, Reed MB, Cowman AF, McFadden GI. Protein trafficking to the plastid
of Plasmodium falciparum is via the secretory pathway. EMBO J 2000;19:1794-
802.
[19] Duraisingh MT, Triglia T, Cowman AF. Negative selection of Plasmodium
falciparum reveals targeted gene deletion by double crossover recombination. Int
J Parasitol 2002;32:81-9.
[20] Maier AG, Braks JA, Waters AP, Cowman AF. Negative selection using yeast
cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum
for targeted gene deletion by double crossover recombination. Molecular &
Biochemical Parasitology 2006;150:118-21.
[21] Wickham ME, Rug M, Ralph SA, et al. Trafficking and assembly of the
cytoadherence complex in Plasmodium falciparum-infected human erythrocytes.
EMBO J 2001;20:1-14.
[22] O'Donnell RA, Freitas-Junior LH, Preiser PR, et al. A genetic screen for improved
plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium
falciparum chromosomes. EMBO J 2002;21:1231-9.
[23] van Dooren GG, Marti M, Tonkin CJ, et al. Development of the endoplasmic
reticulum, mitochondrion and apicoplast during the asexual life cycle of
Plasmodium falciparum. Mol Microbiol 2005;57:405-19.
[24] Tonkin CJ, van Dooren GG, Spurck TP, et al. Localization of organellar proteins in
Plasmodium falciparum using a novel set of transfection vectors and a new
immunofluorescence fixation method. Mol Biochem Parasitol 2004;137:13-21.
[25] Militello KT, Wirth DF. A new reporter gene for transient transfection of
Plasmodium falciparum. Parasitol Res 2003;89:154-7.
[26] Meissner M, Krejany EO, Gilson PR, et al. Tetracycline analogue-regulated
transgene expression in Plasmodium falciparum blood-stages using Toxoplasma
gondii transactivators. Proc Natl Acad Sci U S A 2005;102:2980-5.
[27] Meissner M, Schluter D, Soldati D. Role of Toxoplasma gondii myosin A in
powering parasite gliding and host cell invasion. Science 2002;298:837-40.
[28] Franke-Fayard B, Trueman H, Ramesar J, et al. A Plasmodium berghei reference
line that constitutively expresses GFP at a high level throughout the complete life
cycle. Molecular & Biochemical Parasitology 2004;137:23-33.
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
APPENDIX
Vector Maps
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
Reagents
Bags of red blood cells are obtained from the Red Cross Blood Bank in
anticoagulant citrate phosphate dextrose solution. The red blood cells are
transferred from the bags to sterile bottles for storage at 4°C and are NOT
washed prior to use.
2. Media
For 100 mls of RPMI-Hepes, supplement with 5.8 mls of 3.6% NaHCO3
and 10ml of 5% albumax.
RPMI-1640 10.44 g
25 mM Hepes 5.96 g
200 µM Hypoxanthine 50 mg
H2O 960 ml
Store at 4°C
3. CytoMix
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
6 ml 2M KCl
7.5 µl 2M CaCl2
500 µl 1M MgCl2
To 90 ml with ddH20.
Filter-sterilise.
0.76 g EGTA
To 80 ml with ddH20
4. Pyrimethamine
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5. WR99210
6. Ganciclovir
Working solution: dilute stock 1:10 in H2O (=20mM) (stable for 4 weeks at
4oC)
7. 0.15% Saponin
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
Dissolve 0.15g saponin in 100ml RPMI-Hepes, filter sterilise and store at 4°C
8. 5% Sorbitol
For 500mls:
Autoclave
Dissolve Blocking Reagent 10% (w/v). (bottle 4 of DIG kit) in 1 x Maleic Acid
Buffer at 65°C. Place on stirrer and mix.
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
Thaw 100 x CSPD (vial 5 from DIG kit) when the kit arrives and make 20μl
aliquots. Freeze these aliquots. Avoid repeated freeze/thaw cycles.
A 20μl aliquot diluted 1:100 with Detection Buffer makes up 2mls (enough for
the chemiluminescent detection of a 10 x 10 cm membrane).
6 x SSC, 5 x Denharts,0.1%SDS
17. Saponin
Preparation of medium:
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
19. Heparin
Dissolve the content of 1 ampoule heparin (DBL, 5000 I.U per 1 ml) in 25 ml
RPMI1640 culture medium (pH 7.2) without fetal calf serum
Working solution: Dilute the stock 10 × with dimineralised water and adjust the
pH to 7.2 with 1 M HCl. Autoclave.
21. Nycodenz
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Functional Genomics of Malaria Parasites Workshop Manual – BIOTEC, Bangkok 2007
Selectable Markers
Positive
µl/10ml
selectable Antibiotic Stock solution Working solution Final conc. IC50 Reference:
dish
marker
hDHFR WR99210 20mM in DMSO 20µM in RPMI- 5µl 10nM Fidock and
(Jacobus Hepes Wellems, PNAS
Pharmaceuticals) (4 weeks, 4oC) 1997
Blasticidin Blasticidin S 5mg/ml 5mg/ml 4µl 2µg/ml 150-450ng/ml Mamoun et al.,
hydrochloride in RPMI-Hepes in RPMI-Hepes (strain dep.) PNAS 1999
(Invitrogen) (2 weeks, 4oC,or 8
weeks –20oC, )
Negative
selectable
marker
HsTK Ganciclovir 200mM in H2O 20mM in H2O 10µl 20µM 1.14µM Duraisingh et
(Roche) (3D7) al., IJP 2002
ScCD 5-Fluorocytosine 10mg/ml 1:100 in PBS 5µl 0.38µM Maier et al.,
(Ancotil) 77mM (0.77mM) MBP 2006
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Notes…
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Notes…
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Notes…
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Notes…
63