Gene Targeting in Malaria Parasites

METHODS: A Companion to Methods in Enzymology 13, 148–157 (1997)Article No. ME970507

Gene Targeting in Malaria Parasites

Robert Menard*,1 and Chris Janse†

*Department of Pathology and Department of Medical and Molecular Parasitology, New York UniversityMedical Center, New York, New York 10016; and †Department of Parasitology,University of Leiden, 2300 RC Leiden, The Netherlands

their genome seems to occur only by homologous re-Gene targeting, which permits alteration of a chosen gene combination (1–6). Thus, gene targeting in Plasmo-

in a predetermined way by homologous recombination, is an dium can be achieved with a single (positive) se-emerging technology in malaria research. Soon after the de- lectable marker and does not require negativevelopment of techniques for stable transformation of red markers to counterselect random integration events.blood cell stages of Plasmodium falciparum and Plasmodium Moreover, Plasmodium cells are haploid duringberghei, genes of interest were disrupted in the two species. their entire schizogonic cycle in host erythrocytes,The main limitations of gene targeting in malaria parasites where they can be transformed and cloned. There-result from the intracellular growth and slow replication of fore, the function of proteins encoded by single-copythese parasites. On the other hand, the technology is facili- genes can be studied following single targetingtated by the very high rate of homologous recombination fol- events. Recombination events of various types havelowing transformation with targeting constructs (Ç100%). now been obtained in numerous sites of the Plasmo-Here, we describe (i) the vector design and the type of muta- dium genome (1–6), and targeted blood stages of thetion that may be generated in a target locus, (ii) the selection parasite as well as sporozoites have been character-and screening strategies that can be used to identify clones ized at the phenotypic level (4–6). Gene targeting inwith the desired modification, and (iii) the protocol that was malaria parasites seems on its way to becoming aused for disrupting the circumsporozoite protein (CS) and common tool for studying their biology.thrombospondin-related anonymous protein (TRAP) genes ofP. berghei. q 1997 Academic Press

DESCRIPTION OF METHODS

BackgroundGene targeting, by allowing the introduction of Gene targeting relies on the homologous recombi-specific mutations into virtually any cloned gene, is nation between two copies of a sequence, one presentthe primary tool used to analyze protein function in in the genome (target gene) and the other in thevivo. The power of the technology obviously depends incoming DNA (targeting construct). In its commonon the ease with which targeted clones may be iden- form, the incoming DNA contains (i) the targetingtified, i.e., on the relative frequency of homologous sequence, (ii) a bacterial vector, and (iii) a positiverecombination to its main competing pathway, ran- selectable marker that is used both to select thedom plasmid integration. Plasmodium cells are ideal transfected cells and to mutate the target gene. Twotargets, as integration of incoming constructs into basic configurations of targeting plasmids, replace-

ment and insertion, are commonly used for gene dis-1 To whom correspondence should be addressed. ruption (Fig. 1). The insertion (O-type) construct

148 1046-2023/97 $25.00Copyright q 1997 by Academic Press

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149GENE TARGETING IN MALARIA PARASITES

contains a region of homology to the target gene the wild-type gene. In contrast, the replacement con-struct is expected to integrate via a double crossovercloned as a single continuous sequence, whereas the

replacement (V-type) construct contains two regions between pairs of homologous sequences (Fig. 1, III),leading to the replacement of the target gene by theof homology (the arms of the V) separated by the

selectable marker. The insertion plasmid typically disrupted exogenous copy (allelic exchange). Be-cause no sequence is duplicated in the recombinantintegrates via a single crossover (reciprocal recombi-

nation) between the homologous sequences (Fig. 1, locus, the wild-type gene cannot be recreated.Recombination events in yeasts (7, 8) have longI), leading to the insertion of the entire plasmid into

the target locus. Because the region of homology is provided paradigms for similar events in other eu-karyotes, such as mammalian cells (9, 10), and proto-duplicated in the recombinant locus, the two copies

can recombine to excise the plasmid and regenerate zoan parasites such as Leishmania (11, 12), Trypa-

FIG. 1. Recombinant loci generated after gene targeting with an insertion or a replacement construct. The coding region of thetarget gene (open box) is composed of four internal fragments (1–4) and is flanked by 5* and 3 * untranslated sequences (bold line).The insertion construct is represented here as containing an internal fragment of the gene (2–3) and the replacement construct asa homologous sequence interrupted by the selectable marker (stippled box). Thin lines represent bacterial sequences. The insertionplasmid linearized within the targeting sequence (site B) typically generates direct repeats of the region of homology flanking thecassette and the bacterial sequences (I). Note that if the targeting and targeted sequences are identical (no mutation), then theposition of the actual crossover point between them does not affect the structure of the recombinant allele. Undesired events usuallyconsist of integration of more than one copy of the plasmid arranged in tandem repeats (II), arising by either integration of a constructmultimer or successive rounds of integration. The replacement construct linearized in the plasmid backbone (site Z) or released fromit typically promotes a double crossover leading to the allelic exchange of the wild-type gene by one copy of the mutation (III). Notethat any heterologous sequences at the ends of the targeting regions are not incorporated into the final structure. Complex patternsmay arise from double crossover events involving concatemers of linear molecules (as in IV). The replacement construct in a circular(or recircularized) form can also integrate by a single crossover involving the proximal (V) or distal (VI) part of the targeting sequence.Open arrows point to sites of end-to-end ligation that concatenates or recircularizes the construct. A, B, C, X, Y, and Z symbolizerestriction sites.


150 MENARD AND JANSE

Construct Componentsnosoma (13, 14), and Toxoplasma (15, 16). It is ageneral rule that insertion plasmids recombine more 1. Bacterial vector. A high-copy-number plas-frequently when a double-strand break is introduced mid, such as pUC or pBSKS, should be used to en-into the incoming DNA within its targeting sequence sure high yields of the targeting construct for electro-and that the ends of linear DNA molecules are more poration.recombinogenic (7). Two plasmodial species are now

2. Selectable marker. The selectable markersamenable to gene targeting, the major human patho-available in each plasmodial system are presented ingen Plasmodium falciparum and the rodent parasitethe previous article. The best established selectablePlasmodium berghei. In both Plasmodium systems,marker is the pyrimethamine-resistant variant(s) oftransformation of circular plasmids leads mainly tothe DHFR–TS2 bifunctional gene. Preferably, thethe autonomous replication of the plasmid as an ex-marker should be heterologous to the recipient spe-trachromosomal element. However, in agreementcies, to minimize the risk of competing with the tar-with the ‘‘linear DNA rule,’’ linear constructs readilygeting sequence for homologous recombination. Thisgenerate homologous recombinants in P. berghei. Inis not, however, essential for targeted integration incontrast, it appears that in P. falciparum linear con-any plasmodial system (1, 2). In targeting experi-structs either fail (3) or are less proficient than theirments in P. berghei, we used a cassette containingcircular counterparts (1) in generating homologous5 kb of P. berghei DHFR–TS sequences associatedrecombinants.with shorter targeting sequences (1.5–2.7 kb) andreproducibly obtained resistant parasite populationscomposed exclusively of clones targeted in the geneDesign of Targeting Plasmidsof interest (4, 6). Resistant nontargeted clones may

Replacement or Insertion Plasmid nonetheless arise from replacement/conversion ofthe endogenous DHFR–TS by the incoming mutatedAlthough reports in various eukaryotic systemscopy, as well as from spontaneous mutations in thesuggest that an insertion vector can target the de-endogenous DHFR–TS.sired locus at a higher frequency than a replacement

vector containing the same homologous sequence3. Targeting sequence. In mammalian cells, a(17), the targeting construct most commonly used

single nucleotide mismatch between the targetingfor simple gene disruption is the replacement vector.and targeted sequences can dramatically decreaseOne advantage of the replacement vector is the sta-the frequency of homologous recombination (18, 19).bility of the recombinant locus it creates. However,The importance of sequence divergence to the ratethe possible reversion to the wild-type structure ofof targeting frequency in Plasmodium is unknown.the locus created by plasmid insertion may offer theIn P. berghei, homologous recombination can occuradvantage of complementing a defective phenotype.in the absence of sequence identity between the part-For example, a TRAP knockout was generated byners. For example, targeted integration of a plasmidintegration of an insertion plasmid, and TRAP0 spo-containing a copy of the ribosomal RNA C gene wasrozoites did not infect mosquito and mammalian tar-observed into this gene as well as the D gene, whichget cells. They contained, however, a minor popula-displays 95% identity with the C gene (A. Waters,tion of wild-type TRAP revertants that had regainedpersonal communication). For most applications, itinfectivity, thus demonstrating the role of the muta-is nonetheless preferable that the homologous se-tion in the defective phenotypes (6).quence in the targeting construct originates from theUnlike insertion plasmids, replacement vectorsgenomic DNA of a strain isogenic to the one to becan recombine in a variety of ways (Fig. 1, III–VI).transformed. As shown in other systems, the tar-These include double crossovers involving concate-geting frequency is also likely to increase with themers of linear molecules (as in Fig. 1, IV) and single

crossovers involving circular (or recircularized) mol-ecules, which might not result in a null mutation 2 Abbreviations used: DHFR–TS, dihydrofolate reductase–

thymidylate synthase gene; KAHRP, knob-associated histidine-(Fig. 1, V and VI). To reduce the frequency of theserich protein gene; CS, circumsporozoite protein gene; TRAP,undesired events, replacement constructs are usu-thrombospondin-related anonymous protein gene; PCR, polymer-ally linearized, as a single crossover involving a lin- ase chain reaction; RBC, red blood cell; PBS, phosphate-buffered

ear replacement molecule should lead to a chromo- saline; PRP, parental resistant population; TRP, transfer-resis-tant population.some break.



length of homology. However, considering the rela- inactivated with a replacement construct trans-formed as a linear molecule bearing homologoustively short sequences of homology sufficient for

crossover formation in Plasmodium [as short as ends, which resulted in the expected double cross-overs. TRAP was also mutated using a linearizedÇ500 bp (1, 6)] and the notorious instability of plas-

mid constructs carrying the A/T-rich Plasmodium insertion vector bearing an internal fragment of thegene, which generated two truncated copies ofDNA, stretches of homology should be restricted to

Ç1 kb. To increase the likelihood of generating a TRAP. The markers used (DHFR–TS mutants) orig-inated from Toxoplasma gondii in the KAHRP tar-null mutation after recombination, the replacement

vector is designed to delete part or all of the coding geting construct and from P. berghei in the CS andTRAP targeting constructs.sequence of the target gene. When no deletion is

introduced in the coding region, the selectableTransformation of Linear or Circular Constructs andmarker should be inserted early in the coding se-Selection Methodsquence (or in an upstream exon) of the target gene,

so that if the 5* part of the gene is expressed, a The transformation techniques used in P. falcipa-rum and P. berghei differ in many aspects (see theminimal portion of the wild-type polypeptide is syn-

thesized. Similarly, the insertion plasmid should previous article), particularly with respect to theform of the transformed DNA required for successfulcontain an internal fragment of the target gene so

that both copies in the final structure are inacti- gene targeting and the time required to select thetargeted clones. Transformation with circular plas-vated, one lacking the 5* end and the other the 3 *

end of the gene (Fig. 1, I). mids leads mainly to the replication of autonomousepisomes, in both Plasmodium systems. Episomes

4. Linearization site. Prior to electroporation, are unstably maintained (randomly segregated) andthe replacement vector should be cut either in the lost during cell division if drug pressure is removed.plasmid backbone or at both ends of the homolo- In P. berghei but not P. falciparum, linearization ofgous fragment. The latter case generates a linear the targeting construct is essential for rapid isola-molecule with homologous free ends, which have tion of homologous recombinants. In P. falciparum,been shown in yeasts to recombine with high fre- transformation with circular constructs can none-quencies (20, 21). In Plasmodium, short linker se- theless lead to the selection of homologous recombi-quences of the vector left at one or both ends of the nants, when long selection periods combined withlinear molecule do not impede the correct allelic periods of removal of the drug pressure are appliedexchange (4, 6). The insertion vector must be lin- (1, 3, 5). On average, clonal recombinant parasitesearized within the region of homology, but the re- can be selected in Ç3–4 months in the case of P.striction site used for linearization should not be falciparum (see Fig. 3) and Ç3 weeks in the case oflocated too close to an end of the region of homol- P. berghei (see protocol and Fig. 4). The longer timeogy. This will ensure that exonuclease activity that necessary to select recombinants in P. falciparum isprogresses from the site of linearization does not a consequence of the different transformation tech-eliminate one homologous end, as both are essen- niques employed (linear versus circular constructs,tial for crossover formation. In Plasmodium, inte- transformation into extracellular merozoites versusgration can still occur when an insertion plasmid intraerythrocytic blood stages, and in vivo versusis linearized at a site located 250 bp from one end in vitro selection procedures in P. berghei and P.of the homologous region (6). falciparum, respectively) and the longer generation

cycle of P. falciparum (2 days) than P. berghei (1Examples day). A well-known complication associated with re-

peated erythrocytic cycles of malaria parasites is theFigure 2 shows the targeting constructs that wereused to disrupt the single-copy genes encoding the accumulation of spontaneous genomic rearrange-

ments, including large deletions of protein-encodingknob-associated histidine-rich protein (KAHRP) inP. falciparum (5) and the circumsporozoite protein sequences observed in in vitro-cultured parasites

(22–24). This might complicate the interpretation of(CS) and thrombospondin-related anonymous pro-tein (TRAP) in P. berghei (4, 6). KAHRP was dis- the phenotype of a targeted line. It is thus important

to analyze the phenotype of several clones generatedrupted with a replacement construct transformed asa circular molecule, which promoted a single cross- by independent electroporation experiments. In the

near future, it will be possible with a second se-over and plasmid insertion. CS and TRAP were each



lectable marker to complement the defective pheno- correctly targeted clones, frequently with a singlerestriction digestion. In addition, it can provide antype by restoring the wild-type gene. This is required

to formally demonstrate that the phenotype is due estimation of the proportion of desired recombinantsin the resistant population. In the P. berghei system,to the lack of the target gene product and not to

alteration of expression of another locus. parasites are cloned from the resistant population bylimiting dilution and injected into rats. As a limited

Screening Methods number of clones can be obtained, parasite cloningshould be attempted only if the resistant populationThe presence of the desired recombination eventscontains a sizable proportion of the desired recombi-in the genomic DNA of the resistant population cannation event. A screen should be used that easilybe revealed by PCR or Southern blot analysis.identifies a band diagnostic of the expected event. A

PCR Analysis diagnostic digest of the correctly targeted clone (Fig.1, I or III) can be obtained with a restriction enzymePrimers are chosen that can amplify a specificthat does not cut in the construct, including thejunction fragment following the expected crossoverbackbone of an insertion plasmid (site X): the size ofevent. Typically, one primer is complementary to se-the restriction digest in the correct clone equals thatquences specific to the construct (cassette or bacte-observed in the wild-type population plus the size ofrial sequences) and the second is complementary toone selectable marker (in the case of a replacementsequences of the target locus just beyond the regionconstruct) or of one entire plasmid (in the case of anof homology. Both primers should anneal to se-insertion construct). Following the primary screenquences that are not located more than 2 kb from

one another, which in most cases precludes amplifi- of the resistant population, the clonal alleles mustcation of the entire recombinant locus. Therefore a be subjected to extensive restriction analysis. CarePCR-based screen can hardly be diagnostic of the should be taken to identify undesired recombinationdesired recombination event. events in the target locus. Insertion of multiple cop-

ies of an insertion plasmid (Fig. 1, II) can be detectedSouthern Blot Analysis by digesting the DNA with a restriction enzyme that

cuts the plasmid at a single site in the vector se-In contrast to PCR, Southern blot analysis of theresistant population can in most cases identify the quences (site Z): the presence of a band with a size

FIG. 2. Targeting constructs used to generate KAHRP, CS, and TRAP gene knock-outs. The genomic DNA is represented aboveand the transformed targeting construct below. Coding regions are represented by boxes [hatched, homologous sequences; stippled,DHFR–TS marker from Toxoplasma gondii (KAHRP) or Plasmodium berghei (CS and TRAP)]. Bold lines represent untranslatedregions of the target gene; thin lines represent untranslated regions of the marker, in each case homologous to the recipient species;dashed lines represent bacterial vector sequences. The site of linearization of the TRAP insertion plasmid is indicated by an interrup-tion in the region of homology. The length of homology is given in kilobases, and the selected events are indicated by crosses.



equal to that of the plasmid is diagnostic of multiple pyrimethamine-sensitive and gametocyte-producerplasmid insertion. Similarly, most of the double strain, and is derived from the techniques describedcrossover events involving replacement construct in the previous section (6). Some aspects of the trans-concatemers (Fig. 1, IV) can be recognized by digest- formation and clone selection protocols, however,ing the DNA with a restriction enzyme that cuts in have been optimized for the strain NK65. Gene tar-one region of homology (site A or C): the presence of geting at two distinct genomic loci expressed only ina band of the size of the transformed construct is sporozoites has reproducibly yielded resistant para-diagnostic of multiple unit integration. Simple inser- site populations composed exclusively of the ex-tions of replacement plasmids (Fig. 1, V or VI) are pected recombinants. The selectable cassette con-recognized by probing the DNA with the vector se- sisted of a pyrimethamine-resistant DHFR–TSquences. External probes (annealing to the target mutant of P. berghei expressed by its natural flank-region outside the region of homology) may be used ing regions, borne by plasmid pMD204. Figure 4to analyze the 5* and 3 * ends of the targeted locus, schematically illustrates the various steps of the pro-to ensure that no event has occurred in the locus in tocol.addition to the planned recombination event. For gene targeting experiments involving genes

expressed at posterythrocytic stages of the parasite,GENE TARGETING IN Plasmodium berghei it is important to ensure that targeted clones differ-

entiate into fertile gametocytes. Gametocyte produc-The following protocol is based on gene targeting tion can be rapidly lost on repeated erythrocytic cy-

experiments in P. berghei strain NK65 (4, 6), a cles and parasite transfers to new hosts, and non-gametocyte-producer lines rapidly overgrow thegametocyte-producer population (24). Therefore,both the recipient and the recombinant parasitesshould have gone through a minimal number ofschizogonic cycles, which implies short preelectro-poration and selection periods. The protocol de-scribed below includes Ç30 days of mitotic parasitemultiplications (Ç30 schizogonic cycles) in rat redblood cells (RBCs), starting from the initial sporozo-ite-induced rodent infection up to the time clonalrecombinant parasites can be transferred back tomosquitoes. However, longer protocols involvingÇ60 schizogonic blood cycles still produced recombi-nant populations that infected mosquitoes.

1. Preparation of Recipient Parasites

We use the following schedule, where Day 1 corre-sponds to the day of electroporation.FIG. 3. Gene targeting using pyrimethamine selection in Plas-

modium falciparum. After electroporation, parasites are notDay 010. Salivary glands of infected mosquitoestreated the first 2 days and then treated with pyrimethamine for

Ç4 weeks. After this treatment, the vast majority of the resistant (Anopheles stephensi) at Days 14–18 postfeeding areparasites contain autonomously replicating unrearranged epi- dissected in RPMI 1640 medium (ICN Biomedicals,somes. Parasites are then cultured (i) in the absence of drug pres- Inc., Costa Mesa, CA, Catalog No. 12-609-54). Sporo-sure for 3 weeks (which allows parasites to stop replicating the

zoites released by grinding the glands are separatedplasmid) and again in the presence of pyrimethamine for 4 weeksfrom major cellular debris by centrifugation at 1000g(to select the rare homologous recombinant clones in the popula-

tion of parasites having lost the plasmid), or (ii) in the continuous for 2 min and counted in a hemocytometer, and 4 1presence of small amounts of the drug for 7 weeks. During this 105 in 0.8 ml RPMI 1640 are injected intraperitone-7-week selection period, parasite samples are assessed regularly ally into a hamster.by PCR and/or Southern blot for the presence of the desired ho-mologous recombinants. When the desired clones are detected, Day 04. The parasitemia of the hamster shouldthe resistant population is cloned. The genotype (and phenotype)

be 5 to 10%. The hamster blood is collected by cardiacof the clonal lines can be tested 3 to 4 months after electropora-tion. puncture and diluted in phosphate-buffered saline



(PBS) to obtain 1% parasitemia. Five 150-g Wistar grown overnight in standard Luria–Bertani me-dium. The DNA pellet is resuspended in 10 mMrats are injected intravenously with 0.25 ml of the

diluted blood. Tris–1 mM EDTA (pH 8.0) at a final concentrationof 1–5 mg/ml and digested overnight with restriction

Day 01. The parasitemia of these rats should be enzyme(s) (New England BioLabs, Beverly, MA).between 1 and 3%. Their blood (Ç5 ml each) collected Complete digestion is essential to prevent transfor-by cardiac puncture is cultured separately in vitro mation of circular autonomously replicating plas-for Ç16 h (see previous section). Under these condi- mids. The linearized molecule, or the liberated inserttions, most parasites mature into schizonts that do of a replacement plasmid, may be purified from annot rupture and do not liberate merozoites. agarose gel (Gene Clean, BIO101, Vista, CA) after

0.7% agarose gel electrophoresis, to minimize con-2. Preparation of Targeting Construct tamination with uncut DNA. However, DNA purifi-

cation by phenol–chloroform extraction and ethanolThe targeting plasmid is isolated from Escherichiacoli DH5a using Qiagen Maxiprep (Qiagen Inc. precipitation after complete DNA digestion is also

effective for gene targeting. The DNA is finally re-Chatsworth, CA), starting from 500 ml of bacteria

FIG. 4. Overview of the gene targeting protocol in Plasmodium berghei. Days are represented by numbers, Day 1 being the day ofelectroporation. (1) Preparation of recipient parasites: A hamster is infected by sporozoites extracted from salivary glands of mosquitoesinfected with the pyrimethamine-sensitive parental strain, and 6 days later RBC stages of the parasite are transferred to Wistarrats. After 3 more days of replication in rats, RBC stages are cultured in vitro for 16 h. At the end of the culture, the majority ofRBC stages of the parasite are schizonts. (2) Selection of resistant parasites (based on a construct conferring pyrimethamine resistance):Schizonts/merozoites are electroporated with the targeting construct and injected into highly susceptible young rats. Pyrimethamineis injected once daily, starting 30 h after electroporation and until replicating forms of the parasites are not detected in Giemsa-stained blood smears. A parental resistant population (PRP) rapidly emerges, is treated with pyrimethamine for two consecutivedays, and is transferred to new rats to produce a transfer-resistant population (TRP). The TRP is subsequently cloned by limitingdilution into young rats. Clonal parasite populations usually yield a high parasitemia (ú5%) Ç20–22 days after electroporation. (3)Transfer to mosquitoes: Mosquitoes are then fed on rats infected with clonal recombinant populations as soon as they containgametocytes that exflagellate in vitro. In the case of the wild-type parasites, zygotes/ookinetes (diploid) generate oocysts (polyploid)in the mosquito midgut, from Days 3 to 4 postfeeding. Sporozoites (haploid) are formed inside oocysts from Days 9 to 11 postfeedingand, after being released from oocysts, invade the mosquito salivary glands from Day 14 postfeeding. The entire life cycle of theparasite can thus be completed in Ç45 days.



suspended in sterile water or 10 mM Tris–1 mM solution is prepared by mixing in 20 ml PBS 8 dropsof 1 N HCl, 3 drops of dimethyl sulfoxide (Sigma),EDTA (pH 8.0) at a concentration of 500 ng to 1 mg/

ml. The DNA is stored at 0207C before electropora- and 25 mg of pyrimethamine (Sigma, Catalog No. P-7771), and sonicated for 20 min at room temperaturetion.before injection. Rats are treated for the first time30 h after electroporation (Day 2) and, subsequently,3. Electroporationevery 24 h with a single intraperitoneal injection ofThe techniques for parasite in vitro culture and25 mg/kg pyrimethamine, until replicating forms ofmerozoite collection are described in the previousthe parasite are undetectable on a 15-min examina-section. Briefly, the infected blood in RPMI 1640 me-tion of a Giemsa-stained blood smear (gametocytesdium supplemented with fetal calf serum and hepa-can remain in the blood for longer periods). Thisrin is incubated at 377C for 16 to 18 h under appro-usually occurs at Days 6–8 postelectroporation. Apriate gas conditions. The RBCs infected withparasite population, referred to as the parental re-schizonts are then separated from uninfected cellssistant population (PRP), usually emerges at Dayson density gradients, resuspended in 100 ml PBS8–10 postelectroporation. Parasites are treated with(Dulbecco’s formula without Mg2/ and Ca2/, GIBCO-pyrimethamine for 2 consecutive days as describedBRL, Catalog No. 14190), and immediately used forabove, starting at the day of their detection (para-electropration. The parasites collected from one do-sitemia ú0.01%). We frequently obtained resistantnor rat, i.e., from one culture flask, constitute thepopulations that expanded slowly (regardless of py-recipient parasites of one electroporation experi-rimethamine treatment), perhaps because of the de-ment. The quality of the in vitro culture may varyvelopment in rats of an antiparasite immune re-considerably from one flask to another. We performsponse.experiments in duplicate and choose the cultures

After the 2-day drug treatment, parasites of thehaving the highest schizont/gametocyte ratios at thePRP are passaged to a naive rat, yielding a transfer-end of the culture. A high ratio (ú3) is usually associ-resistant population (TRP). Parasites should beated with a high parasitemia at Day 1 (4 h) postelec-transferred by intravenous injection to induce a par-troporation (ú0.2%) and is our best predictive valueasitemia in the recipient rat(s) of 0.01–0.1% 2 daysof successful gene targeting.after transfer. The rat harboring the PRP is thenAfter schizont collection, 10 to 50 mg of linear DNAsacrificed immediately after transfer, and the geno-(usually in less than 30 ml) is mixed to a final volumemic DNA of the PRP subjected to Southern blot anal-of 300 ml with PBS, and with the merozoites originat-ysis to assess the presence of the correctly targeteding from one flask (Ç109 in 100 ml). The mixture (400clones and evaluate their proportion. The parasites

ml) is then transferred into a prechilled electropora-in the TRP may then be cloned when the rat para-tion cuvette (0.2-cm electrode gap, Catalog No. 165-sitemia is between 0.1 and 1%, if at least Ç10% of2086, Bio-Rad) and subjected to an electric pulsethe parasites contain the desired modification.with a Bio-Rad apparatus (800 V, 25 mF), usually

yielding a time constant of 1.3 { 2 ms. The electro-poration cuvette is then immediately placed on ice, 5. Parasite Cloningand after 4 min, 0.2 ml of electroporated parasites is

Parasite cloning by limiting dilution should be per-injected into the tail vein of each of two anesthesizedformed from blood containing õ1% infected RBCs,young (Ç21-day-old, Ç80-g) Sprague–Dawley rats.and with less than 10% of infected RBCs containingmore than one parasite. The parasitemia and the

4. Parasite Selection number of RBCs per microliter of blood of the donorrat are counted, and the blood is diluted in 20% fetalThe parasitemia of the recipient rats is checked

by Giemsa staining of a drop of blood obtained from calf serum–RPMI 1640 to a dilution containingõ0.1parasite/ml. Rats are then injected in the tail veinthe tail of the animals, 4 h after electroporation and

daily thereafter. Four hours after electroporation, with a blood dilution supposed to contain 0.5 para-site in 0.2 ml PBS. To maximize the likelihood ofyoung trophozoites (ring forms) should be visible in

0.2 to 1% of the RBCs. The typical selection scheme injecting not more than one parasite, we considervalid only the cloning experiments that infect notis outlined in Fig. 4.

Rats are treated daily by intraperitoneal injection more than 4 of 10 animals injected. When clonalpopulations have infected 1% of the RBCs and gener-of pyrimethamine at 25 mg/kg body weight. The drug



ate mature gametocytes that readily exflagellate on is proven only by restoring the wild-type phenotypewith the wild-type gene. This requires a second (posi-a microscope slide, mosquitoes can be fed on rats for

at least 2 consecutive days and/or the rat blood can tive) selectable marker. Such a marker would alsooffer a way to study genes essential for the erythro-be collected by cardiac puncture, and the parasite

genomic DNA analyzed by Southern blotting. cytic cycle of the parasite, which needs to be unal-tered for selecting recombinant lines. For example,parasites diploid for the essential target gene could6. Preparation of Parasite Genomic DNAbe created with a copy carried by an (removable)

The blood of the animal having a parasitemiaú1% episome, and the genomic copy subsequently dis-is collected by cardiac puncture, mixed with an equal rupted. A second selectable marker would also allowvolume of PBS, and filtered through a Plasmodipur double gene disruptions or introduction of a modifiedfilter (Eurodiagnostica), which retains white blood version of a gene in a disruptant background.cells. RBCs are then pelleted by centrifugation Genetic modifications induced by a marker inser-(300g, 10 min) at room temperature and washed tion are generally restricted to gene inactivation.twice in 20 ml PBS, and the RBC pellet (Ç3–4 ml) Their main disadvantage is that a selectable markeris resuspended in twice its volume of 0.1% saponin in the final locus not only disrupts a sequence but(Sigma) in PBS. The solution is mixed vigorously may also alter expression of closely linked genesand left for 10 min at room temperature for RBC with its own control elements (26) or even alter ex-lysis to proceed. The freed parasites are then pel- pression of distant loci (27). It is therefore preferableleted (1000g, 10 min, in 1.5-ml Eppendorf tubes), to obtain mutations with no exogenous sequence leftand washed in PBS the number of times necessary in the locus, especially when subtle modifications,to obtain a perfectly clear supernatant. Parasites such as single-base-pair substitutions, are intro-from one infected animal are then incubated at 507C duced in the target gene. This can be achieved within 600 ml lysis buffer (10 mM Tris, pH 7.6, 50 mM a number of systems, such as the ‘‘hit and run’’ andEDTA, pH 8.0, 0.1% sodium dodecyl sulfate, protein- ‘‘tag and exchange’’ procedures [for a review, see Ref.ase K 100 mg/ml) at least 2 h, preferably overnight. (28)], which consist of introducing the mutation withThe parasite DNA is then recovered by two phenol– a positive selectable marker and removing all exoge-chloroform extractions followed by one chloroform nous sequences with a negative selectable markerextraction, precipitated in 100% ethanol, washed to leave only the subtle modification. Such negativetwice in 70% ethanol, air-dried for 10 min, and fi- selectable markers, which are yet to be developed innally resuspended in 50–100 ml 10 mM Tris-1 mM plasmodial systems, would constitute an importantEDTA (pH 8.0) containing RNase. tool in gaining a precise understanding of malarial

protein function.

CONCLUDING REMARKSACKNOWLEDGMENTS

Perhaps the most encouraging aspect of our stilllimited experience with gene targeting in Plasmo- We thank Victor Nussenzweig and Andy Waters for their help-dium parasites is the clear-cut phenotype of the ful comments and critical review of the manuscript.knock-out parasites that have been generated. Vitalsteps of the parasite life cycle and infectivity appearto depend on unique pathways, and disruption of asingle gene can block the life cycle of these parasites. REFERENCESIt can thus be hoped that the genetic analysis ofother important Plasmodium molecules will greatly 1. Wu, Y., Kirkman, L. A., and Wellems, T. E. (1996) Proc. Natlhelp in elucidating their function. Few additional Acad. Sci. USA 93, 1130–1134.tools are still missing, however, for precise in vivo 2. Van Dijk, M. R., Janse, C. J., and Waters, A. P. (1996) Sciencestructure–function analysis. 271, 662–665.

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Gene Targeting in Malaria Parasites