EUKARYOTIC CELL, Apr. 2009, p. 530–539
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 8, No. 4
Tagging of Endogenous Genes in a Toxoplasma gondii Strain
My-Hang Huynh and Vern B. Carruthers*
Department of Microbiology and Immunology, University of Michigan School of Medicine,
1150 W. Medical Center Dr., Ann Arbor, Michigan 48109
Received 31 October 2008/Accepted 2 February 2009
As with other organisms with a completed genome sequence, opportunities for performing large-scale
studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a
system for tagging genes endogenously with yellow fluorescent protein (YFP) in a ?ku80 strain. Ku80 is
involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have
shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homol-
ogous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-
thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette,
which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that
the ?ku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus
than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic
screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known
micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion.
We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our
findings demonstrate that the combination of the ?ku80 strain and the pYFP.LIC constructs reduces both the
time and cost required to determine localization of a new gene of interest. This should allow the opportunity
for performing larger-scale studies of novel T. gondii genes.
Toxoplasma gondii is an obligate intracellular protozoan in
the phylum Apicomplexa that has garnered more intense study
in recent decades. This is due in part to the genetic tractability
and ease of growth of T. gondii and also because knowledge
obtained for this parasite is often relevant to its kin, including
other pathogens of medical and veterinary importance, such as
Plasmodium falciparum, the parasite that causes malaria (11).
The genome sequence of Toxoplasma was recently completed
(10), opening the door to identifying novel genes involved in a
variety of events, such as cell invasion, replication, gliding
motility, metabolism, stage conversion, and virulence. With a
large number of new genes of interest, it is essential to have
tools that enable investigators to perform studies on a larger
scale. One bottleneck for the analysis of novel genes is protein
localization, since this generally necessitates time-consuming
production of antibodies that often require additional affinity
purification. Protein localization by ectopic expression of a
tagged construct can also be problematic due to overexpres-
sion or mistiming of expression from a heterologous promoter.
If genes could be tagged directly on the chromosome, this
would better mimic natural expression and allow the use of
fluorescent protein tags or standardized antibodies to monitor
DNA double-strand breaks in mammalian cells are repaired
via either the homologous recombination or nonhomologous
end-joining (NHEJ) pathways, which differ in the requirement
for homologous sequences versus ligation independent of
DNA sequence homology. DNA breaks can be lethal to an
organism if left unrepaired, resulting in genomic instability,
sensitivity to DNA damage, and mutations leading to tumor
development (16). Ku70 and Ku80 (Ku86 in higher eu-
karyotes) proteins form a heterodimeric complex that plays a
pivotal role in NHEJ DNA repair. Ku70 and Ku80 orthologues
are widely present in eukaryotes, having been identified in
yeast, plants, invertebrates, and vertebrates (5). While the pri-
mary role of Ku proteins is in DNA repair, they have also been
implicated in a range of other cellular activities, such as telo-
mere maintenance, tumor suppression, gene transcription reg-
ulation, heat shock-induced responses, and apoptosis (15, 43).
As observed by in vitro photo cross-linking of the Ku dimer
and DNA, the Ku70 subunit preferentially contacts the major
groove and Ku80 contacts the minor groove (48). Crystallog-
raphy studies show that the Ku70/80 complex is in a 1:1 ratio in
an open ring-shaped structure through which a DNA helix can
pass (45). In a model of NHEJ, the Ku complex, which has a
high affinity for DNA, binds to DNA termini and recruits the
DNA-dependent protein kinase catalytic subunit, activating its
kinase activity. This is followed by the recruitment of the DNA
ligase IV and XRCC4 complex to the double-strand break,
resulting in ligation of the DNA ends (5).
Neurospora crassa mutants deficient in Ku70 or Ku80
showed 100% homologous recombination, in contrast to the
wild-type strain, which displayed only 21% homologous recom-
bination (25). Deletions of either Ku70 or Ku80 in the fungal
strain Sordaria macrospora or in Aspergillus spp. (3, 30, 40)
have resulted in considerable improvements in gene targeting
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, University of Michigan School of Medicine,
1150 W. Medical Center Dr., Ann Arbor, MI 48109. Phone: (734)
763-1928. Fax: (734) 764-3562. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://ec
?Published ahead of print on 13 February 2009.
without notable growth impairment or other defects. The data
on the effect of Ku70 or Ku80 disruption in mice or cell lines
on susceptibility to DNA damaging agents are conflicting and
unclear. Several studies have claimed an increased rate of
mutation, chromosomal instability, and increased carcinogen-
esis (7, 12, 20, 26, 41), while other investigators observed a
converse decrease in mutation frequency and chromosomal
rearrangements (32, 44). It appears that the particular mouse
strain or cell line used may play a role in this discrepancy.
We sought to exploit the potential improvement in homol-
ogous recombination in a T. gondii Ku80-disrupted strain to
introduce a reporter protein at the 3? end of a gene in order to
localize the product within the parasite. We found that the
Ku80-null parasite strain significantly improved gene targeting
efficiency compared to wild-type parasites and is a useful tool
for endogenous gene tagging. In a companion article, Fox,
Bzik, and colleagues also show the utility of ?ku80 parasites for
creating targeted gene knockouts (9).
MATERIALS AND METHODS
Fusion PCR knockout (KO) construct for disruption of Ku80. Genomic flank-
ing sequences of the Ku80 gene (583.m05492) were obtained from the Toxodb
database (www.toxodb.org; version 4.3). Primers were designed to amplify ?4 kb
of the 5? and 3? ends flanking the gene. The flanks were amplified to overlap on
one end with the hypoxanthine xanthine guanine phosphoribosyltransferase
(HXGPRT)-selectable marker cassette, and similarly, the HXGPRT marker
cassette was amplified to contain Ku80 sequences on the ends (Fig. 1A).
Primers to amplify the 3? flank were P-1, 5?-GTCGACATGCATATGTTTT
AGAGG-3?, and P-2, 5?-CCGCGGGCGGGTTTGAATGCAAGGTTTCGTG
CTGcatgagtcgatatatctcgctagctataaatatatc-3? (lowercase indicates HXGPRT se-
quences and uppercase indicates Ku80 gene sequences). Primers to amplify the
5?flank were P-3, 5?-gttctggcaggctacagtgacaccgcggtggGATACTCACTGTGGGT
TGAGTTACAAG-3?, and P-4, 5?-ATGTAACTGTGCGCCTATCTACTTC-3?.
Primers to amplify HXGPRT were P-5, 5?-GATATATTTATAGCTAGCGAG
ATATATCGACTCATGcagcacgaaaccttgcattcaaacccgcccgcgg-3?, and P-6, 5?-CT
RH genomic DNA was used as template for amplifying the 5? and 3? flanks,
and pminiHXGPRT plasmid (6) was used as a template for amplifying the
HXGPRT selectable marker. A fusion PCR product was then amplified using
primers P1 and P4 and the three individual 5?, 3?, and HXGPRT fragments as
?ku80 transfection and selection. The RHhxgprt?(6) strain was cultured by
passage in human foreskin fibroblasts in Dulbecco’s modified Eagle’s medium–
HEPES–10% fetal bovine serum at 37°C and with 5% CO2. Parasites were filter
purified, pelleted, and resuspended in cytomix buffer (2 mM EDTA, 120 mM
KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM HEPES, 5 mM
MgCl2? 6H2O; pH 7.6). Fifteen ?g of the 3?Ku80-HXGPRT-5?Ku80 fusion PCR
product was electroporated into 1 ? 107RHhxgprt?parasites using a Bio-Rad
X-Cell electroporator (settings of 1.5kV, 25 mF, and no resistance). After over-
night growth, transformants were placed under selection with 25 ?g/ml myco-
phenolic acid and 50 ?g/ml xanthine. Transformant pools were tested for the
presence of a KO before cloning by limiting dilution under drug selection.
Individual KO clones were replated in 96-well plates to ensure clonality. This
strain was termed ?ku80-HXG and is the strain used in all the experiments
except those in Fig. 3D, below, which also included the ?ku80-DHFR strain,
which was generated by replacement of Ku80 with the dihydrofolate reductase-
thymidylate synthase (DHFR-TS) selectable marker in RHhxgprt?parasites by
using pyrimethamine selection.
Confirmation of ?ku80 by PCR. Primers within the HXGPRT selectable
marker cassette, dhfr 5? end 3.R (5?-GCGGGCGGGTTTGAATGCAAGGT
TTCGTGC-3?) and outside the 3? genomic flanking region, 3?Ku80.2730891.F (5?-C
ATACCTTCGAGTTGCTCTTCTTGTTGAC-3?), were used to detect the re-
placement of the Ku80 gene with HXGPRT. The absence of the Ku80 gene was
confirmed with primers TgKu80.949.F (5?-TTCCTGATCCCCGTGTATGT-3?)
and TgKu80.1748.R (5?-GACGCCGGATTGTAGTGTCT-3?); the presence of
the HXGPRT selectable marker was confirmed with the primers TgKu80-5?
dhfrHXGPRTdhfr-70bp.F (5?- GATATATTTATAGCTAGCGAGATATATC
-3?) and 5?Ku80.2740775-3?dhfr.R (5?- CTTGTAACTCAACCCACAGTGAGT
Southern blot analysis. Digoxigenin (Roche)-labeled probes against HXGPRT
were amplified from pminiHXGPRT using primers HXG.661.F (5?-GAGAAC
TTACTTCGGCGAG-3?) and HXG.978.R (5?-ATCGACTTCGGACCGACG-
3?) and against Ku80 from genomic DNA using TgKu80.949.F (5?-TTCCTGAT
CCCCGTGTATGT-3?) and TgKu80.1748.R (5?-GACGCCGGATTGTAGTGT
CT-3?. Genomic DNA was digested with XmnI or SacII for probing with
HXGPRT and with XmnI or PstI for probing with Ku80.
pYFP.LIC.HXG and pYFP.LIC.DHFR vectors. The YFP and DHFR-TS 3?-
untranslated region (UTR) fragment was PCR amplified from the plasmid
pTubYFPYFP-sagCAT (13), with a ligation-independent cloning (LIC) cassette
5? to the start of YFP, and subcloned with KpnI and XhoI restriction enzyme
ends into the pminiHXGPRT plasmid (6), generating the pYFP.LIC.HXG vec-
tor. The LIC cassette was introduced into the 5? end of the YFP (35), with the
FIG. 1. Targeted deletion of Ku80. (A) Generation of the Ku80 KO construct. Ku80 5? and 3? genomic flanks were amplified with overlaps to
the dhfr-HXGPRT-dhfr selectable marker; the dhfr-HXGPRT-dhfr marker was amplified with overlaps to Ku80 flanks. A fusion PCR ?ku80
product consisting of Ku80 flanks and the selectable marker was amplified. (B) Primer sets were used to evaluate the parental strain (RHhxgprt?)
and ?ku80 for replacement of the Ku80 gene with the selectable marker (a and a?), the presence or absence of the Ku80 gene (b and b?), and the
presence or absence of the HXGPRT selectable marker (c and c?).
VOL. 8, 2009ENDOGENOUS TAGGING OF T. GONDII GENES 531
following modifications: the LIC cassette (5?- CTGTACTTCCAATCCAATTT
AATTAAAATTGGAAGTGGAGGACGG-3?) contains a unique PacI site, un-
derlined in the sequence, and the stop codon in the original LIC cassette was
removed to allow readthrough to the YFP. The HXGPRT cassette was then
excised with HindIII and NotI and replaced with the DHFR-TS selectable
marker cassette. The vector is a pBluescript backbone, and selection in Esche-
richia coli was performed with 100 ?g/ml ampicillin.
Gene amplification, cloning, and transfection. Genomic sequences were ob-
tained from the Toxodb database (version 4.3) and primers were designed to
amplify 1 to 4 kb of the 3? ends of genes, which contained a unique restriction
enzyme within the fragment. All forward primers contained the LIC sequence
5?-TACTTCCAATCCAATTTAATGC-3? and all reverse primers contained the
LIC sequence 5?-TCCTCCACTTCCAATTTTAGC-3?. The additional GC was
added to the reverse primers to allow amplification of genes up to the second-
to-last codon and for subsequent T4 DNA polymerase and dCTP treatment.
Since a C is necessary for this treatment, this allowed the same LIC sequence to
be used for all genes. In addition, this kept the YFP in frame with the gene after
“ligation.” All reverse primers stopped just prior to the stop codon to allow
readthrough to the downstream YFP. Both the pYFP.LIC.DHFR vector and the
PCR products were treated with T4 DNA polymerase as described previously
(35). Briefly, 0.2 pmol of PCR product was incubated in a 20-?l reaction mixture
with 5 mM dithiothreitol (DTT), 4 mM dCTP, 1? T4 DNA polymerase buffer,
and T4 DNA ligase and the following condition: 30 min at 22°C, 20 min at 75°C,
and cooling on ice. Two ?g of linearized vector DNA was incubated in a similar
reaction mixture (60 ?l), with dGTP in place of dCTP. Two ?l of T4-treated PCR
product and 1 ?l of vector were incubated for 10 min at room temperature, 1 ?l
of 25 mM EDTA was added, and the mixture was incubated for another 5 min
at room temperature before placing it on ice. Electrocompetent E. coli cells were
transformed with 1 ?l of reaction mix. Positive transformants were identified and
confirmed by diagnostic restriction enzyme digests. For transfection, 2 to 20 ?g
of constructs was linearized within the region of homology, phenol-chloroform
extracted, and ethanol precipitated. Nicked/open circle constructs were prepared
by overnight digestion with EcoRI in the presence of 500 ?g/ml of ethidium
bromide at 26°C, followed by two phenol-chloroform and two chloroform-
isoamyl alcohol extractions, as described previously (29). After overnight growth,
transformants were selected with 2 ?M pyrimethamine and kept under selection
until the experiment was terminated.
Fluorescence and immunoblot analysis. For localization, egressed parasites
were inoculated onto eight-well chamber slides and fixed in 4% paraformalde-
hyde 24 h postinfection. YFP fluorescence was viewed directly on a Zeiss Axio
inverted microscope with a YFP filter cube. For colocalization immunofluores-
cence, inoculated slides were fixed, permeabilized with 0.1% Triton X-100,
stained with rabbit anti-MIC2 (1:250) (47) and mouse anti-green fluorescent
protein (anti-GFP; 1:250; Clontech/BD) antibodies, followed by Alexa594-con-
jugated goat anti-rabbit and Alexa488-conjugated goat anti-mouse secondary
antibodies (Invitrogen). 4?,6-diamidino-2-phenylindol (DAPI; 5 ?g/ml final con-
centration) was added to visualize the nuclei.
For immunoblot analyses, parasites were isolated and lysed in 95°C sample
buffer and separated on sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis gels. After semidry transfer to polyvinylidene difluoride membranes, blots
were blocked, probed with mouse anti-GFP (1:1,000), washed, and probed with
goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson
Plaque assay. For transfection followed by plaque assay, 10 ?g aliquots of
linearized acyl carrier protein (ACP), microneme protein 3 (MIC3), and prolif-
erating cell nuclear antigen (pCNA) in pYFP.LIC.DHFR were prepared and
transfected into ?ku80-HXG. Transfected parasites were diluted and 750
tachyzoites were inoculated into D150 petri dishes with pyrimethamine selection
and left undisturbed for 7 days. Individual plaques were picked and expanded in
96-well and then 24-well plates, genomic DNA for PCR was extracted using
Qiagen DNEasy columns, and proper integration into target loci was visualized by
fluorescence. Integration of the construct into the endogenous DHFR-TS locus was
detected using primers in the first and second exons of dhfr-ts: dhfr-ts.5?.268.F,
5?-GTTTCCTTTTTCTTCTGTTCGTTTC-3?, and dhfr-ts.896.R, 5?-GAATCCTT
Generation of a ?ku80 strain. Ku80 is a molecule found in
a heterodimer with Ku70, and together they are involved in
DNA repair via the NHEJ pathway. Deletion of Ku80 should
result exclusively in homologous recombination, increasing the
efficiency of genetic knockouts and incorporation of reporter
proteins to endogenous loci. BLAST searches of the Toxo-
plasma genome database (version 4.3) using the Neurospora
crassa Ku70 and Ku80 genes (mus-51 and mus-52; GenBank
accession numbers AB177394 and AB177395, respectively) in-
dicated the presence of single orthologues of Ku70 and Ku80 in
T. gondii (with BLASTp values [Toxodb accession numbers] as
follows: 2.6 ? 1013[50.m03211] and 4.9 ? 107[583.m05492],
respectively). Both TgKu80 and TgKu70 have a canonical so-
called Ku78 domain, which is the hallmark of this protein
family. We focused on genetically disrupting TgKu80, using
a fusion PCR-based method to replace the Ku80 gene with a
selectable marker. To create the Ku80 KO (?ku80) strain, a
fusion PCR KO construct was generated, consisting of 3?Ku80
and 5?Ku80 genomic flanks fused on either side of the HXGPRT
selectable marker cassette (Fig. 1A). Once the individual PCR
products were amplified, all three fragments were used as
templates in a fusion PCR that resulted in a 3?Ku80-HXGPRT-
5?Ku80 product. This KO construct was transfected into
RHhxgprt?parasites and KO clones were identified by PCR
using a forward primer annealing outside the 3? flank of the
KO construct and a reverse primer within the HXGPRT cas-
sette. Clones were also tested by PCR for the absence of the
Ku80 gene and the presence of the HXGPRT cassette. Paren-
tal RHhxgprt?parasites were included as a control. The re-
sults from one representative ?ku80 clone are shown in Fig.
1B. Southern blot analysis was performed to confirm the pres-
ence of a single copy of the selectable marker and the absence
of the Ku80 gene (data not shown). A similar method was used
to generate a ?ku80 strain with pyrimethamine selection in
RHhxgprt?parasites (data not shown).
Construction of the pYFP.LIC.DHFR vector and LIC
method. To improve the efficiency of cloning individual genes
into an expression vector, a plasmid was created that contained
a LIC cassette (1, 35) upstream of an in-frame copy of YFP
and a DHFR-TS selectable marker cassette (Fig. 2A). For
insertion into the YFP vector, genes were amplified with prim-
ers that introduced LIC sequences at the 5? and 3? ends of the
PCR products, homologous to the LIC sequences upstream of
YFP. Restriction enzyme digestion with PacI linearized the
vector, and subsequent T4 DNA polymerase treatment of both
the vector and the PCR product exposed compatible sequences
that annealed and were subsequently ligated after transforma-
tion of E. coli. All amplified genes were inserted into the YFP
vector with this LIC method.
To exploit ?ku80 parasites for gene tagging, we amplified
the 3? genomic region of a gene up to but not including the
stop codon and cloned this fragment into the pYFP.LIC.DHFR
vector, creating a fusion of the gene-of-interest fragment with
YFP. By reciprocal recombination, the region of homology in
the construct recombined with the homologous sequences on the
chromosomal gene, introducing the YFP to the 3? end of the
endogenous gene (Fig. 2B). Studies in the yeast model have
demonstrated that a plasmid linearized within a region of homol-
ogy to chromosomal DNA increases the efficiency of homologous
integration 10- to 1,000-fold; these and other studies formed the
basis for the double-strand break repair model of recombination
(27, 28, 39). In this model, the 3? ends are processed to form 3?
single-stranded tails, which then invade the homologous duplex
and priming DNA synthesis (review in reference 38). Because
532 HUYNH AND CARRUTHERSEUKARYOT. CELL
only a fragment (the 3? region) of each gene is used for targeting
and the YFP does not contain its own promoter for expression,
YFP fluorescence in the expected pattern should only be seen
upon homologous targeting and correct integration into the en-
dogenous locus. For proof of principle, several genes that were
previously tagged with reporter proteins and shown to target
properly were examined: pCNA (31), ACP (46), MIC3 (37), and
targeted correctly to their respective organelles. Immunofluores-
cence dual staining was performed with anti-MIC2 antibody to
visualize the apical end of the parasite or with ROP2 to localize
It is worth noting that ACP and pCNA are essential genes
(14, 46), indicating that chromosomal YFP tagging of these
proteins did not overtly interfere with function. Having estab-
lished that the YFP vector can successfully target an endoge-
nous gene, we sought to determine whether the form of the
transfected construct, i.e., supercoiled, linear, or nicked/open
circle, affects the efficiency of integration. Using ACP and
pCNA as easily identifiable YFP-tagged proteins in the para-
sites, we prepared constructs in all three forms (Fig. 3A). As
expected based on the double-strand break model, only con-
structs linearized within the region of homology integrated into
the target loci, while the supercoiled or nicked plasmids never
integrated during the 35-day observation period with py-
rimethamine selection (Fig. 3B). We also transfected con-
structs that were linearized downstream of the DHFR-TS se-
lectable marker cassette with NotI (Fig. 2A) and did not
observe integration after 30 days (data not shown). Given these
results, it is necessary to amplify a region of the 3? end of the
gene that contains a unique restriction enzyme site to linearize
the construct after cloning in the YFP vector. Next we exam-
ined whether the site of linearization affects integration. ACP-
YFP was used since it contains two unique sites within the 1-kb
targeting region and tagged ACP is easily identified within the
parasite. Digests cutting 350 bp or 750 bp from the 3? end of
the ACP targeting sequence were transfected into parasites
and examined by fluorescence for 33 days to determine the
efficiency of integration (Fig. 3C). No apparent difference in
the time required for integration of the two constructs was
observed, indicating that linearization as close as 350 bp from
the 3? end of the gene of interest is sufficient for targeting if
there are homologous sequences upstream of this cut site. The
amount of transfected vector DNA required was also tested,
and no difference was observed using 2.5 ?g or 20 ?g of DNA
(data not shown).
To directly compare the efficiency of homologous targeting
in ?ku80 and wild-type parasites, as well as mycophenolic
acid/xanthine (MPA/X) versus pyrimethamine selection, we
transfected ACP, pCNA, and MIC3 in pYFP.LIC.DHFR into
?ku80-HXG and RH strains and ACP, pCNA, and MIC3 in
pYFP.LIC.HXG in ?ku80-DHFR and RH and examined in-
tegration of the constructs over time by fluorescence. pCNA-
YFP-positive parasites reached 90% of the population by 13
days posttransfection (dpt) in the ?ku80-HXG strain and 40%
in the ?ku80-DHFR strain; both reached 100% within ?20 dpt
(Fig. 3D). In the RH strain, pCNA-YFP.DHFR-positive fluo-
rescent parasites emerged much more slowly, reaching 12% by
18 dpt and ?70% by 34 dpt. It is possible that given sufficient
time in selection, all parasites would be fluorescent with trans-
fection of this particular gene. In contrast, no fluorescent par-
asites were observed with pCNA-YFP.HXG in RH. ACP-
YFP-positive parasites were detected in ?ku80-HXG at 11 dpt
and reached 100% by 27 dpt. ACP-YFP parasites in ?ku80-
FIG. 2. Endogenous gene tagging. (A) A pYFP.LIC.DHFR vector was generated that bore an upstream LIC cassette for cloning of genes of
interest fused in frame to YFP, which was followed by the DHFR-TS 3? UTR and a DHFR-TS cassette for selection of resistant parasites.
(B) Schematic illustration of the single-crossover mechanism of integration of the YFP fusion protein to the 3? end of a gene. (C) Positive control
genes ACP (apicoplast), pCNA (nucleus), MIC3 (micronemes), and ROP1 (rhoptries) were cloned into the YFP expression vector and transfected
into parasites, and localization was visualized by immunofluorescence with a GFP antibody. The DAPI staining in the ACP panel is pseudocolored
with cyan to bring out the staining in the apicoplast. MIC2 staining was used to identify the micronemes and apical end of the parasite, and ROP2
staining was used to identify the rhoptries.
VOL. 8, 2009 ENDOGENOUS TAGGING OF T. GONDII GENES533
DHFR were slower to emerge and reached ?60% by 34 dpt. In
RH, however, no ACP-YFP-positive fluorescent parasites were
observed with the MPA/X selection, but a small percentage
(4%) of YFP-positive parasites were observed in the py-
rimethamine selection 34 dpt. Interestingly, MIC3-YFP-posi-
tive parasites reached ?13% in ?ku80-DHFR by 20 dpt but
then dropped to 3% by 23 dpt and to only ?1% by 34 dpt. No
fluorescence was observed in the ?ku80-HXG or RH transfec-
tions through the duration of the experiment (34 dpt). This
indicates that (i) different genes have varied receptiveness to
recombination and tagging, (ii) the ?ku80 strain is more ame-
nable to homologous recombination and gene tagging than the
wild-type RH strain, and (iii) pyrimethamine selection appears
to work better than MPA/X selection in some but not all cases.
To utilize the ?ku80 strain and YFP tagging to investigate
novel genes, we compiled a list of genes based on several
criteria: (i) identification in a MudPIT/2DE proteomic screen
of excreted-secreted antigen (ESA) products (49); (ii) evi-
dence of a cell cycle expression profile consistent with a role in
parasite invasion (M. Behnke and M. White, personal commu-
nication); (iii) the presence of domains previously identified in
microneme proteins; and (iv) the presence of mass spectros-
copy peptides in the Toxoplasma genome database. We present
six examples of tagged genes that were confirmed to be ex-
pressed by both immunoblot analysis and by fluorescence. Ta-
ble 1 shows all the genes tested, including the gene IDs, the
predicted molecular masses of the proteins, and the localiza-
tion of the protein by YFP. All primers used to amplify tested
genes are provided in the Table S1 of the supplemental mate-
rial. After cloning the genes into pYFP.LIC.DHFR and trans-
fection/selection, transformant pools were examined for YFP
expression. While expression of some YFP could be viewed
directly by microscopy, many required staining with anti-GFP
antibody to enhance the signal. The overall success rate of this
method as measured by positive YFP signal after transfection
and selection was 74%.
Perforin-like protein 1 (PLP1) is a novel microneme protein
identified in a MudPIT analysis (49) that contains a MACPF
pore-forming domain (18). PLP1 is predicted to be a 113-kDa
protein (Table 1), although it migrates at 130 kDa on reducing
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In
PLP1-YFP parasites, the PLP1 band shifted up to ?160 kDa
(Fig. 4B), suggesting correct integration of the tagging con-
struct at the PLP1 locus. Accordingly, antibodies against PLP1
and GFP showed an identical pattern, consistent with mi-
croneme staining (Fig. 4A, panel i).
The 72.m00001 gene is annotated as a 62-kDa poly(ADP-
ribose) glycohydrolase (PARG) family protein. YFP tagging of
this protein showed localization in the parasitophorous vacuole
and, in particular, an overlap with the intravacuolar membra-
nous nanotubular network (42), as revealed by colocalization
with the dense granule protein GRA2 (24) (Fig. 4A, panel ii).
A single band at ?80 kDa was observed by immunoblotting
(Fig. 4B), consistent with the expected size of the YFP chi-
Gene 49.m03355 is a hypothetical protein with a predicted
molecular mass of 12 kDa. After endogenous tagging,
49.m03355-YFP lysates showed a single band at ?38 kDa (Fig.
4B). This protein localized to a distinct spot at the anterior
pole of the tachyzoite near where the conoid is positioned (Fig.
FIG. 3. Optimization and efficiency of gene tagging. (A) Agarose gel showing plasmids in the forms supercoiled (SC), linearized within the
targeting sequence (LN), or nicked/open circle (OC). (B) Linearization of the vector is necessary for efficient integration. Only the LN construct
integrated into the homologous loci, whereas the SC and OC constructs did not integrate during the 30-day experiment. (C) Linearization site and
efficiency of integration. Two restriction enzyme digestion sites were used, cutting 350 or 750 into the 1-kb targeting sequence. Both linearized
constructs were shown to be equally effective in integration into the endogenous ACP locus. (D) ?ku80 favors homologous recombination.
pCNA-YFP, ACP-YFP, and MIC3-YFP constructs with either HXGPRT or DHFR-TS selectable markers were transfected into RH or ?ku80,
and YFP-positive parasites were enumerated over time and are expressed as a percentage of the total population. Note that the y-axis scale of the
MIC3-YFP graph is smaller than for the pCNA-YFP and ACP-YFP graphs.
534 HUYNH AND CARRUTHERSEUKARYOT. CELL
4A, panel iii). 49.m03355-YFP localized anterior to the inner
membrane complex (IMC), as visualized with an antibody
against IMC1. In dividing parasites, a discrete 49.m03355-YFP
signal was detected just anterior of the developing IMC of the
daughter cells (Fig. 4A, panel iii?). Rotation of a three-dimen-
sional image obtained by optical sectioning of 49.m03355-YFP
parasites revealed that the signal corresponded to a ring-like
structure, possibly one of the polar rings associated with the
conoid (Fig. 4A, panel iii, inset). Since this was an exception-
ally small gene, the entire genomic fragment, including the
endogenous promoter and upstream sequences, was amplified
to include a unique restriction site. Thus, in this particular
case, we cannot exclude the possibility that this construct in-
tegrated randomly and is expressing the YFP-tagged version
ectopically as an extra copy of this gene.
83.m00006 is a hypothetical protein with similarity to Plas-
modium falciparum SPATR (secreted protein with altered
thrombospondin type I repeat domain), which is localized to
the surface of sporozoites and the apical region of asexual
erythrocytic stages (4). 83.m00006 was identified in a pro-
teomic analysis of Ca2?-dependent secretion (19) and is pre-
dicted to encode a 58-kDa protein. A single ?95-kDa band was
observed in 83.m00006-YFP lysates (Fig. 4B). 83.m00006-YFP
localized to the apical region, where it partially overlapped
with MIC2 staining, indicating that it may reside to some
extent in the micronemes (Fig. 4A, panel iv).
49.m00054 showed punctate staining slightly posterior to the
micronemes (Fig. 4A, panel v). In colocalizations with
proM2AP, which occupies a site termed the VP1 compartment
(16a), a fraction of 49.m00054 appeared to colocalize with
proM2AP at certain stages of replication (data not shown). In
other vacuoles, the 49.m00054-YFP signal was found posterior
to proM2AP (data not shown). The predicted molecular mass
of 49.m00054 is 101 kDa, and by immunoblot analysis using an
anti-GFP antibody two bands were observed, at ?100 kDa and
?135 to 140 kDa (Fig. 4B). The higher molecular mass band is
consistent with the addition of a 27-kDa YFP tag; the lower
band may represent a processed form. No bands were observed
in an induced ESA fraction (data not shown), suggesting that
the tagged protein is likely not secreted.
20.m03858 is a hypothetical protein with no orthologues or
putative conserved domains. It is highly expressed in RH (www
.toxodb.org) and is predicted to be a 97-kDa transmembrane
protein. 20.m03858-YFP is localized to the apical or subapical
region of the parasite; some colocalization with MIC2 is ob-
served (Fig. 4A, panel vi). A band of 130 kDa was seen by
TABLE 1. Genes chosen for YFP tagging of ?ku80
Cathepsin protease L
Waller et al. (46)
Guerini et al. (14)
Ossorio et al. (28a)
Zhou et al. (49)
Zhou et al. (49)
Zhou et al. (49)
Zhou et al. (49)
Toursel et al. (41a)
Mann et al. (23)
Kasper et al. (18a)
Putative MIC maturase
MVEc/lysosome Anterior to nucleus
Anterior to nucleus
Non-TM Ag ?poly(ADP-ribose)
C2 domain protein
Cathepsin protease B
Hypothetical (25 EGFd)
Zhou et al. (49)
Kawase et al. (19)
Invasion cell cycle
Zhou et al. (49)
Zhou et al. (49)
Invasion cell cycle
Invasion cell cycle
Invasion cell cycle
Que et al. (30a)
Zhou et al. (49)
Invasion cell cycle
aAs of October 2008.
bPeptide(s) identified by mass spectroscopy according to Toxodb.
cMVE, multivesicular endosome.
dEGF, epidermal growth factor.
eAlso known as Apple domains.
fNA, not applicable.
gPV, parasitophorous vacuole.
hGenes 8.m00176 and 8.m00178.
VOL. 8, 2009 ENDOGENOUS TAGGING OF T. GONDII GENES535
immunoblotting, implying integration of the YFP in the en-
Completion of the Toxoplasma genome sequencing project
and the availability of the Toxoplasma genome database un-
leashes the possibilities of studying large cohorts of genes pre-
dicted to play roles in particular cellular events. While gene
disruption can be a powerful approach for determining protein
function, deciphering the subcellular localization of a protein
can also provide important clues to its role in a process or
pathway. We present here a combination of approaches for
improving the efficiency of determining protein localization.
First, we generated a T. gondii strain lacking Ku80, which
functions with Ku70 in NHEJ DNA repair. In the absence of
Ku80, homologous recombination predominates. We have ex-
ploited this to introduce a YFP tag to genes at their endoge-
nous loci. We also generated a YFP expression construct con-
taining a LIC cassette for convenient cloning of genes of
interest. The LIC method of cloning proved much more effec-
tive and efficient than conventional restriction enzyme-medi-
ated subcloning, since a single restriction digest of the vector
allows the cloning of any insert with complementary LIC se-
quences. This makes it an ideal method for high-throughput
cloning of inserts.
Several proteins that have previously been tagged with GFP
were used as positive controls for testing the effectiveness of
this construct and system. ACP, pCNA, MIC3, and ROP1 all
FIG. 4. Localization of novel gene products by endogenous gene tagging. (A) YFP-tagged genes by immunofluorescence. (i) Microneme
protein PLP1. (ii) 72.m00001, poly(ADP-ribose) glycohydrolase. (iii and iii?) 49.m03355 hypothetical protein. Arrowheads point to the YFP signal
in apical poles of developing daughter cells. Inset: ring-like localization pattern. (iv) 83.m00006, SPATR-like protein. (v) 49.m00054, dystroglycan
domain protein. (vi) 20.m03858 hypothetical protein. Scale bars were generated within the Zeiss Axio program. (B) Immunoblot of YFP-tagged
genes probed with anti-GFP. Positions of molecular mass markers (in kDa) are indicated.
536 HUYNH AND CARRUTHERSEUKARYOT. CELL
targeted properly to their respective organelles and, impor-
tantly, this analysis showed that essential genes, such as ACP
and pCNA, are amenable to tagging. However, IMC1-YFP
and HSP60-YFP failed to show expression, even after multiple
attempts. One potential explanation is that the YFP is cleaved
from the gene product, a likely scenario with IMC1 after the
protein is incorporated into the inner membrane complex (23).
Thus, a limitation of the system is that tagging and visualiza-
tion of gene products that undergo C-terminal proteolysis or
glycophosphatidyl inositol anchor addition are not feasible.
Homologous recombination generally requires either a dou-
ble-strand break in one of the two DNA double helices (plas-
mid or genomic DNA) or a single-strand nick in both helices
(2, 22). Early studies performed in yeast determined that plas-
mids containing a double-strand break in the region of homol-
ogy to genomic sequences recombined with high efficiency
after yeast transformation (28). Linearization of the integrat-
ing construct results in ends-in recombination, wherein the
entire plasmid integrates into the genome at the region of
homology and creates a duplication of the homologous se-
quence; a chromosomal break does not occur because the
break or gap is repaired by gene conversion (reviewed in ref-
erence 38). In the absence of construct linearization, proper
integration is dependent on the low probability of a chromo-
somal break at the targeted locus. After analyzing constructs in
supercoiled, linear, or nicked/open circle forms, we confirmed
that only constructs linearized within the gene fragment inte-
grated into the homologous locus. Linearization of the con-
struct near the selectable marker did not result in proper
integration of YFP into the gene of interest, but it is possible
that a proportion of these pyrimethamine-resistant parasites
contained the DHFR-TS selectable cassette in the endogenous
DHFR-TS locus by double-crossover gene replacement. This
would result in YFP-negative, pyrimethamine-resistant para-
sites. Based on the findings of Fohl and Roos, however, these
parasites have a fitness defect compared to those with either a
normal copy or those with exogenous copies in addition to the
endogenous gene (8). Thus, such parasites may only be seen in
situations where tagging of the target locus results in a sub-
stantial growth defect.
Linearization within the gene fragment requires the pres-
ence of a unique restriction site, which may necessitate ampli-
fication of a larger fragment of DNA to include such a site.
Fortunately, this was not a hindrance, since we found that LIC
cloning of larger fragments (3 to 4kb) was equally efficient as
that with shorter lengths (1 to 2 kb). In the case of 49.m03355,
a particularly small gene of 240 bp, an additional 2.7 kb of
upstream genomic DNA was amplified to include a unique
restriction enzyme site. No other genes were present in the
additional DNA amplified, but since the targeting sequence
inserts by homologous recombination, any gene(s) present
would nonetheless be reconstituted after integration.
The positive control YFP-tagged proteins targeted properly
to their expected organelles, and many tagged hypothetical
proteins showed a pattern consistent with being in apical in-
vasion organelles (rhoptries and micronemes). However, addi-
tional genes selected for tagging either never showed YFP
expression (55.m04618, 8.m00176, 8.m00178 80.m02161, and
50.m00008) or displayed a pattern that resembled that of
mistargeting or retention (20.m03958 and 49.m00054) in a
subapical location, which is presumably a staging ground for
the apical invasion organelles (Table 1). Similar results have
been seen with some exogenously GFP-tagged genes encoding
secretory proteins (49). These gene products do not appear to
have a feature in common, e.g., a transmembrane anchor,
which could account for the failure of the fusion protein to
reach the invasion organelles. Bands detected on immunoblots
were the predicted sizes for the YFP-tagged proteins, implying
that the proper genes were tagged. There are several possible
scenarios: (i) these proteins may naturally occupy a subapical
site; (ii) the precursor is observed in a subapical compartment
and the YFP is cleaved from the mature protein prior to
continuing on to the invasion organelles (as the case may be for
49.m00054); or (iii) the YFP tag hinders the protein from
moving through the secretory pathway to its final destination.
Distinguishing among the possibilities would require produc-
ing antibodies to establish the normal location of the protein.
We are currently generating and testing additional constructs
with smaller epitope tags, which may better facilitate the cor-
rect localization of the tagged gene product.
The genes chosen for tagging were based on their potential
role in invasion, identified by the similarity of their expression
profiles to other known invasion proteins or by their presence
in an ESA proteomic screen. While we expected most of these
genes to be in the invasion organelles, there were exceptions.
For example, the 49.m03355 protein showed a distinct signal at
the extreme apical tip of the parasite, with no overlap with
microneme proteins AMA1 or MIC2 or with the inner mem-
brane complex protein IMC1. The pattern is similar to Tg-
CAM1 and TgCAM2 (17) which, like 49.m03355, are observed
at the apical tip of mature parasites and in developing daughter
cells. The resolution of light microscopy is insufficient to as-
certain whether 49.m03355 is localized to the apical polar rings
or the microtubules comprising the conoid body.
In addition to the YFP vector, we have also engineered con-
structs containing mCherry, tandem dimer Tomato (tdTomato),
and cyan fluorescent reporter proteins (21, 33, 34). We spec-
ulate that low pH in the apical invasion organelles may hamper
the YFP signal, and assessing additional fluorescent reporter
proteins may improve signal detection. Since pCNA-mCherry
and pCNA-tdTomato constructs integrated properly and are
correctly expressed in the nucleus (data not shown), these
vectors can now be used to try tagging genes that did not show
fluorescence with YFP. Constructs encoding YFP and cyan
fluorescent protein may also be utilized in fluorescent reso-
nance energy transfer interaction studies of two chromosom-
ally tagged proteins. Another application of the tagging
method is to identify interacting partners in protein complexes.
To this end, we have generated a tagging construct with a tandem
affinity purification tag for isolation of protein complexes. The
combination of these constructs, the LIC cloning method, and the
?ku80 parasite strains, should prove to be useful tools for more
rapid determination of protein localization and interacting part-
ner proteins for the functional analyses of novel proteins.
This work was supported by a grant from the U.S. National Institutes
of Health (AI046675 to V.B.C.).
We are grateful to Michael Behnke and Michael White for sharing
data prior to publication. We thank Tracey Schultz for technical as-
VOL. 8, 2009ENDOGENOUS TAGGING OF T. GONDII GENES537
sistance and members of the Carruthers lab for helpful discussions,
and we also thank David Bzik for constructive exchanges of ideas and
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