trypanosoma brucei: a first-generation cre-loxp site-specific recombination system
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Experimental Parasitology 106 (2004) 37–44
www.elsevier.com/locate/yexpr
Trypanosoma brucei: a first-generation CRE-loxPsite-specific recombination system
Brian Barrett, Douglas J. LaCount, and John E. Donelson*
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA
Received 14 July 2003; accepted 5 January 2004
Abstract
The bacteriophage CRE-loxP system of DNA recombination is widely used to manipulate segments of the genomes of mice and
other eukaryotes for the purpose of studying the regulation and functions of their genes. Since this recombination system could have
similar applications in analyzing the genomes of trypanosomatids, we assessed the action of CRE recombinase on its loxP DNA
recognition sites in Trypanosoma brucei after inserting tetracycline-regulated CRE and two 34-bp loxP sites into the T. brucei ge-
nome. We found that when loxP sites flank in a direct orientation the transcription termination sequence (1.1 kb) of the T. brucei
GPEET/PAG3 locus, CRE recombinase deletes this termination sequence, permitting transcription and subsequent expression of a
downstream reporter gene for the green fluorescent protein (GFP). Thus, the CRE-loxP system is highly efficient in T. brucei, but the
experimental results also indicate that a better way than the existing tetracycline-regulated system is required to completely silence
expression of CRE in the T. brucei genome when it is not needed before the full range of CRE-loxP applications currently used in
mice can be exploited in African trypanosomes.
� 2004 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: Trypanosoma brucei; CRE recombinase; Procyclin; Transcription termination; Gene deletion; GPEET, glycine–
proline–glutamic acid–glutamic acid–threonine pentapeptide repeat protein (one of the two forms of the protein previously called PARP or
procyclin); EP, glutamic acid–proline dipeptide repeat protein (one of the two forms of the protein previously called PARP or procyclin); PAG,
procyclin-associated gene; TERMGPEET , the transcription terminator sequence of the GPEET/PAG3 locus of T. brucei; FACS, fluorescent activated
cell sorter; GFP, green fluorescent protein; Otet, tetracycline operator; PGPEET and PrRNA, promoters for the T. brucei GPEET/PAG3 locus and rRNA
gene locus, respectively; PT7, promoter for T7 RNA polymerase; rRNA, ribosomal RNA; UTR, untranslated region; VSG, variant surface glyco-
protein
1. Introduction
CRE recombinase is a protein encoded by bacterio-
phage P1 that mediates recombination between two
non-tandem 34-bp loxP sites. Depending on the orien-tation and location of the two loxP sites, CRE can
mediate DNA deletions, inversions, and cross-overs.
Through the pioneering work of Brian Sauer and
colleagues, the technology of this prokaryotic recombi-
nation system has been transferred to eukaryotic cells—
first to yeast cells (Sauer, 1987) and subsequently to
mammalian cells (Sauer and Henderson, 1988). The
CRE-loxP system now forms the basis of many elegant
* Corresponding author. Fax: 1-319-335-9570.
E-mail address: john-donelson@uiowa.edu (J.E. Donelson).
0014-4894/$ - see front matter � 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2004.01.004
approaches for genomic DNA modifications and gene
rearrangements in mice, including conditional gene
knockouts (reviewed by Le and Sauer, 2001; Mills and
Bradley, 2001; Wilson and Kola, 2001). We decided to
introduce this DNA recombination system into the Af-rican trypanosome, Trypanosoma brucei, in anticipation
that it could be used for purposes as diverse as: (i)
forcing recombination between different VSG expression
sites, (ii) deleting large tandem arrays of homologous
genes such as the heat shock protein (HSP) or MSP
(GP63 zinc metalloprotease) gene families, (iii) deleting
or inverting large polycistronic, non-homologous gene
clusters, (iv) targeting different foreign DNA insertionsto the same genomic location so that positional effects
are eliminated during comparison of different gene ex-
pression cassettes, and (v) removing excess selectable
markers for reuse in subsequent constructs.
38 B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44
To test the CRE-loxP system in T. brucei we utilizedthe gene for the green fluorescent protein (GFP) and a
transcription termination sequence located downstream
of the GPEET/PAG3 gene cluster of T. brucei that was
identified and characterized by Pays and colleagues
(Berberof et al., 1996). This gene locus was previously
called the PARP A locus (PARP, procyclic acidic re-
petitive protein or procyclin), but in accordance with the
need for a standardized nomenclature of trypano-somatid genes (Clayton et al., 1998), it was renamed
GPEET/PAG3 (Roditi and Clayton, 1999). The
GPEET/PAG3 transcription terminator sequence (ab-
breviated here as TERMGPEET ) has been shown to cause
an orientation-dependent termination of transcription
from promoters for the GPEET/PAG3 genes, the rRNA
genes and the VSG gene expression sites, all of which are
promoters for either RNA polymerase I or an enzymebearing RNA polymerase I-like activity with respect
to inhibition by a-amanitin (Berberof et al., 1996). This
TERMGPEET does not terminate transcription by
T. brucei RNA polymerase II (Berberof et al., 1996).
We first cloned theCRE coding sequence in a T. brucei
expression vector so that it was under the control of two
adjacent tetracycline operators (Otet). When this vector
was integrated into the genome of T. brucei cells engi-neered to possess the tetracycline repressor protein (pro-
cyclicT. brucei 29–13 cells;Wirtz et al., 1999), we expected
CRE expression to be repressed until it was induced by
addition of tetracycline to the culture medium. Next we
generated a PCR amplification product that contains a
1.1-kb TERMGPEET sequence flanked by direct repeats of
the 34-bp loxP sites and cloned this PCRproduct between
the promoter for the GPEET/PAG3 locus (PGPEET ) andGFP in another T. brucei plasmid. We anticipated that
when this second plasmidwas integrated into theT. brucei
29–13 genome containing CRE, expression of GFP from
the PGPEET would be prevented by the presence of the
intervening TERMGPEET until this terminator sequence
was excised by the activity of CRE acting on the loxP sites
flanking it. Thus, the functional presence of the CRE-
loxP system in T. brucei could be assayed by a ‘‘gain ofactivity,’’ i.e., the gain ofGFP expression, which is amore
convincing demonstration of a targeted genomic DNA
rearrangement event than the loss of an activity. We
found: (i) byWestern blot that CRE expression in theseT.
brucei cells was induced by tetracycline, and (ii) by se-
quence determination of PCR products from the cells
which acquired green fluorescence that CRE did indeed
trigger deletion of the TERMGPEET between the directloxP repeats. However, even when CRE expression is
suppressed by two upstream tandem Otet elements, it
appears that a few CRE molecules are made, which can-
not be detected on a Western blot but are sufficient to
catalyze a deletion of the 1.1-kb segment between two
loxP sites. Therefore, the CRE-loxP system is clearly very
efficient in T. brucei but a better method of completely
inhibiting expression of CRE until its activity is neededmust be identified before the full power of the CRE-loxP
system can be exploited to manipulate the trypanosome
genome inways similar to its current use formanipulating
other eukaryotic genomes.
2. Materials and methods
2.1. Trypanosome cells
The procyclic Trypanosoma brucei brucei cell line 29–
13, which constitutively expresses T7 RNA polymerase
and the tetracycline repressor (Wirtz et al., 1999), was
provided by G.A.M. Cross. Procyclic cells were cultured
in Cunningham�s SM medium supplemented with 20%
heat-inactivated fetal calf serum, 12 lgG418/ml and 25 lghygromycin/ml, and were stably transfected with plas-
mids via electroporation as described previously (Hill
et al., 1999). When the cells were grown in the presence
of additional antibiotics, the following concentrations
were used: phleomycin (2.5 lg/ml) and noursothricin
(100 lg/ml).
2.2. Construction of plasmids
Plasmid pLEW100 was the gift of G.A.M. Cross. The
luciferase coding region (LUC) was excised from
pLEW100 by cleavage withHindIII and BamHI, and the
recessed 30 ends filled in by a DNA polymerase reaction
and ligated together in a DNA ligase reaction to generate
the circular plasmid pLEW100/DLUC (plasmid A in
Fig. 2). The CRE coding sequence (GenBank AccessionNo. X03453) was PCR-amplified from plasmid pBS185
(purchased from Gibco-BRL/Life Technologies, Carls-
bad,CA) using a forward primerwith a 50HindIII site and
a reverse primer with a 50 BamHI site. The resulting PCR
product of HindIII-CRE-BamHI was cloned into Hin-
dIII-/BamHI-digested pLEW100 so that CRE replaced
LUC to generate plasmid pLEW100/CRETi (plasmid B in
Fig. 2).Plasmid pSK1-1/loxP (TERMGPEET )loxP-GFP (plas-
mid D in Fig. 2) was generated using several DNA cas-
settes and plasmids as follows.ClaI- andHindIII-digested
pXS2-pac (plasmid 1, provided by J. Bangs and described
in Bangs et al., 1996) was used as the basic parent plasmid
in which to clone the PCR product of ClaI–loxP(-
TERMGPEET )loxP–HindIII (cassette 1) to generate plas-
mid 2. The primers used to generate cassette 1 are showninFig. 1.AscI–GFP-30-50ACT–AscI (cassette 2)was PCR-
amplified from pHD496:GFP (plasmid 3, described in
Hill et al., 2000) and ligated into AscI-digested plasmid 2
to generate plasmid 4. The fragment HindIII–SAT–
BamHI (cassette 3), which confers resistance to nour-
seothricin via streptothricin acetyltransferase (SAT), was
PCR-amplified from pIR1–SAT (plasmid 5, Joshi et al.,
Fig. 2. Diagrams of plasmids used in this work. Plasmid A is pLEW100 (Wirtz et al., 1999) in which the luciferase coding region (LUC; not shown)
between the indicatedHindIII and BamHI sites was removed by cleavage with these two enzymes followed by a DNA polymerase reaction to ‘‘fill-in’’
the ends and a blunt-end ligation reaction to regenerate a circular plasmid. Plasmid B is pLEW100 in which the LUC coding region between the
HindIII and BamHI sites was replaced with a PCR-amplification product of the CRE coding region. Plasmids A and B were linearized with NotI and
individually electroporated into procyclic T. brucei 29–13 cells (which contain the genes for the tetracycline repressor and T7 RNA polymerase; Wirtz
et al., 1999), selecting for integration into the rRNA gene locus by expression of phleomycin (bleomycin, BLE) resistance. Phleomycin-resistant
clones of each of these transfections were obtained and used in the subsequent experiments. Plasmids C and D were constructed as described in
Section 2. Plasmid D is identical to plasmid C except that it possesses a 1.1-kb TERMGPEET flanked by the 34-bp loxP sites inserted between the
indicated ClaI and HindIII sites of plasmid C. This [loxP(TERMGPEET )loxP] segment was generated using the PCR primers shown in Fig. 1.
Plasmids C and D were linearized with NotI and individually electroporated into cloned procyclic T. brucei 29–13 cells containing either plasmid A or
plasmid B already integrated into their genome. Linearized plasmids C and D integrate into the intergenic region of the b-tubulin gene locus and cells
containing the integrated plasmids were selected for by resistance to nourseothricin (SAT). The puromycin-resistance gene (PUR) shown in the
diagrams of plasmids C and D is a component of the parent plasmid, but was not used in these experiments. PT7 is the bacteriophage promoter
recognized by T7 RNA polymerase. PGPEET and PrRNA are the promoters for the T. brucei GPEET/PAG3 locus and the rRNA genes, respectively.
Otet is the tetracycline operator. The indicated regions flanking the various genes depicted by the rectangles are the corresponding 30 or 50 flankingregions of the following T. brucei genes: 30 and 50 ACT (actin gene), 50 EP (EP gene), 30 ALD (aldolase gene) and 30 TUB (tubulin gene). Dotted lines
indicate the locations of insertions used to generate plasmids B and D. The loxP sites in plasmid D are indicated by the open horizontal arrows.
Abbreviations for restriction sites are indicated in the upper right. The PCR primers used to generate the data shown in Fig. 4A are indicated by the
circled letters F and R above plasmid C, and their sequences are shown on the bottom right.
Fig. 1. Sequences of the forward (top sequence) and reverse (bottom sequence) PCR primers used to amplify a 1.1-kb GPEET/PAG3 terminator
sequence (TERMGPEET ) from the T. brucei genome. The boxed regions are the 34-bp loxP sequence, which consists of two 13-bp inverted repeats (the
CRE recognition sites, indicated by horizontal arrows) separated by an 8-bp asymmetrical spacer that is responsible for the directional nature of the
loxP site (Le and Sauer, 2001; Sauer, 1987). The 50 ends of the primers contain restriction sites for ClaI or HindIII and the 30 ends are the sequencesflanking a T. brucei TERMGPEET (Berberof et al., 1996).
B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44 39
1995) and ligated into HindIII–BamHI-digested plasmid
3. PCR amplification was then used to amplify XhoI-
rRNA promoter-50EP-SAT-30ACT-XhoI (cassette 4),
which was then ligated into XhoI-digested plasmid 4 (to
generate plasmid 6). The resultantKpnI–NotI fragment of
plasmid 6 (cassette 5) was excised and ligated into KpnI-/
NotI-digested pSK1 (described in LaCount et al., 2002) to
produce pSK1-1/loxP (TERMGPEET )loxP-GFP (plasmid
D in Fig. 2). pSK1-1/GFP (plasmid C in Fig. 2) is plasmid
D without cassette 1.
40 B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44
2.3. Flow cytometric analysis
Analiquot of 106 T. brucei cells was centrifuged at 800g
for 5min and the cell pellet gently suspended in 1ml of
a PBS solution containing 0.25% paraformaldehyde.
Green fluorescence was analyzed on a fluorescence
activated cell sorter (FACScan from Becton–Dickinson,
San Jose, CA) at the Flow Cytometry Facility of the
University of Iowa.
2.4. Other procedures
PCR amplifications were conducted for 30 cycles of
the following temperature changes: 95 �C for 45 s, 55 �Cfor 60 s, and 72 �C for 90 s. After the final cycle the re-
action was incubated at 72 �C for an additional 10min
and placed at 4 �C until applied to a 1% agarose gel.Western blots were conducted as described (Hill et al.,
2000) and were probed with a 1–30,000 dilution of
polyclonal rabbit anti-CRE antiserum (Novagen, Mad-
ison, WI) and a 1–7500 dilution of donkey anti-rabbit
secondary antiserum linked to horse radish peroxidase
(Amersham Biosciences, Piscataway, NJ). The relative
amounts of CRE protein were determined by densi-
tometry of the Western blot signals.
Fig. 3. A plot of the appearance of CRE recombinase in extracts of
procyclic T. brucei 29–13 cells containing integrated plasmid B bearing
CRE (see Fig. 2), as detected by Western blots, after induction with the
following concentrations of tetracycline: 1lg/ml (open circles), 0.1 lg/ml (closed circles) or 0.01lg/ml (closed triangles). Cell extracts were
prepared prior to tetracycline addition (0 time) and 1, 2, 4, and 8 h
after tetracycline addition. The insert shows an example of a Western
blot containing extracts of cells induced with 1 lg tetracycline/ml and
probed with anti-CRE antiserum.
3. Results
3.1. Expression of CRE in T. brucei procyclic cells
CRE recombinase has been widely used in a number of
eukaryotic cells to catalyze the excision or inversion of a‘‘floxed’’ DNA segment, i.e., a DNA segment flanked by
the 34-bp loxP elements. If the two flanking loxP ele-
ments are in a direct orientation, CRE catalyzes the de-
letion of the floxed DNA segment, leaving a single loxP
site. If the flanking loxP elements are in an inverted ori-
entation, CRE inverts the DNA segment, rather than
excising it. Furthermore, a single loxP element in the
genome can serve as a target site for insertion of exoge-nously plasmid DNAs containing a loxP element, greatly
enhancing the chances that different transfected plasmids
always insert at the same genomic location (Le and Sauer,
2001; Mills and Bradley, 2001; Wilson and Kola, 2001).
To examine whether CRE can be expressed in T.
brucei, the CRE coding sequence was PCR-amplified
from a commercially available plasmid (see Section 2)
and used to replace the luciferase coding region in T.
brucei expression plasmid pLEW100 (Wirtz et al., 1999).
This new plasmid, pLEW100/CRETi (plasmid B in
Fig. 2), contains PGPEET followed by two adjacent Otet
elements and the CRE coding region. The plasmid was
linearized with NotI, which cleaves once in the plasmid�srRNA spacer sequence, and electroporated into T.
brucei procyclic 29–13 cells, selecting for phleomycin
resistance. T. brucei 29–13 cells are engineered to con-stitutively express the tetracycline repressor (as well as
T7 RNA polymerase) (Wirtz et al., 1999), so we antici-
pated that in the absence of tetracycline, transcription
from PGPEET would be blocked at the two Otet elements
and CRE would not be expressed until tetracycline was
added to the procyclic culture medium. The transfected
T. brucei cells were cloned by serial dilution, and several
clones were selected for growth in liquid culture andfurther study. PCR amplifications of the genomic DNAs
from these clones revealed that the linearized plasmid
had integrated into the rRNA locus (not shown). Clones
of T. brucei 29–13 cells containing integrated pLEW100/
DLUC (plasmid A in Fig. 2) were also generated for use
in subsequent experiments as control cells lacking CRE
(see below).
A clone of T. brucei 29–13 cells containing integratedplasmid B (pLEW100/CRETi) was grown to mid-loga-
rithmic stage (3� 106 cells/ml) in the absence of tetra-
cycline and the culture divided into aliquots to which
differing amounts of tetracycline were added. Samples
were taken at subsequent time points and cell extracts
prepared to determine the amount of CRE protein via
Western blots probed with anti-CRE antiserum. As
shown in Fig. 3, CRE protein could not be detectedprior to tetracycline addition, but was present 1 h after
addition and continued to increase in amount for at
least 8 h. The extent of CRE induction was identical for
both 1 and 0.1 lg tetracycline/ml, but no detectible in-
duction occurred in 0.01 lg/ml. This result and the ki-
netics of induction are consistent with an earlier analysis
of tetracycline induction of this expression system
using luciferase as a reporter gene, in which 1 and 0.1 lg
B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44 41
tetracycline/ml gave similar levels of induction and0.01 lg/ml gave much less (Wirtz et al., 1999).
During these experiments two additional observa-
tions were made concerning the growth of the cells in
culture. First, in the absence of tetracycline, T. brucei
29–13 cells containing plasmid B consistently grew
about 10% slower than did the same cells containing
either pLEW100 or pLEW100/DLUC (plasmid A in
Fig. 2) integrated at the same rRNA locus. This resultsuggests that, even though it cannot be detected on a
Western blot (Fig. 3), a low level of CRE expression
may occur in the absence in tetracycline that retards cell
growth and is not blocked by the existence of two Otet
elements immediately preceding CRE. Secondly, after
adding either 1 or 0.1 lg tetracycline/ml, the growth rate
of cells containing plasmid B diminished still further, the
cells stopped growing by 24 h and they eventually died.In contrast, tetracycline had no effect on the growth of
T. brucei 29–13 cells containing integrated pLEW100 or
plasmid A. Likewise, when tetracycline was removed
after 1 h from cells bearing plasmid B by extensively
washing the cells with medium without tetracycline and
then re-suspending them in tetracycline-free medium at
their former concentration, the cells returned to their
previous growth rate. Thus, prolonged induction ofCRE appears to be deleterious to the cells.
3.2. Detection of CRE recombinase activity at loxP sites
introduced into the T. brucei genome
Having generated tetracycline-inducible CRE re-
combinase in T. brucei, we now wanted to establish an
assay for CRE-catalyzed DNA recombination in T.
brucei that would result in the acquisition, rather than
loss, of an activity since this would be a positive dem-
onstration of the recombination event, rather than a
negative one. Thus, we constructed a plasmid bearing
GFP preceded by a cassette containing the 1.1-kb
TERMGPEET sequence flanked by directly oriented loxP
elements, i.e., plasmid pSK1-1/loxP (TERMGPEET )loxP-
GFP (plasmid D in Fig. 2). The recombination assaystrategy was based on the fact that initially transcription
from PGPEET in this integrated plasmid should be ter-
minated by TERMGPEET before it reaches GFP. Then,
after CRE-catalyzed excision of TERMGPEET , tran-
scription through GFP should occur, resulting in GFP
expression that can be readily detected visually.
The T. brucei 29–13 cells containing CRE in inte-
grated plasmid B are resistant to neomycin, hygromycin,and phleomycin, so it was necessary to build into plas-
mid D a fourth antibiotic resistance gene that is not
downstream of TERMGPEET . Thus, plasmid D has a
cassette bearing the SAT gene (nourseothricin resis-
tance) under the control of PrRNA, and the PUR gene
(puromycin resistance) downstream of the terminator
sequence in the plasmid is not used. Plasmid C in Fig. 2
is a control plasmid in which the cassette of [loxP(TERMGPEET )loxP] in plasmid D has been removed so
that transcription can proceed directly from PGPEET to
GFP. Plasmids C and D are designed to integrate into
the intergenic region of the TUB locus when linearized
with NotI.
Linearized plasmids C and D were transfected into
T. brucei 29–13 cells containing either previously inte-
grated plasmid A or B, and cloned transfectant cells wereselected by resistance to nourseothricin. As expected, in
the absence of tetracycline, cells containing either inte-
grated plasmid A or B initially fluoresced green when
transfected with plasmid C and did not when transfected
with plasmidD (not shown). This result is consistent with,
and confirms, the original finding of Pays and colleagues
that the TERMGPEET region diminishes transcription
from PGPEET by 7–20-fold (Berberof et al., 1996), anddemonstrates that the flanking loxP elements do not in-
terfere with this termination of transcription.
After several days in liquid culture, however, a few
cells in the cloned populations containing plasmids B
and D began to fluoresce green, even though tetracycline
was not present in the culture medium. In contrast,
green fluorescent cells did not appear in the cloned
populations containing plasmids A and D. The per-centage of cells in the cloned populations containing
plasmids B and D that fluoresced green varied some-
what from one population to another, but in all cases
this percentage of green cells increased with continued
passage of the cells in culture. These results are consis-
tent with the possibility suggested above that a low level
of CRE expression occurs from integrated plasmid B,
even in the absence of tetracycline.To test this possibility, PCR amplifications of the re-
gion surrounding the cassette of [loxP(TERMGPEET )
loxP] were conducted on genomic DNA templates iso-
lated from several cloned populations bearing different
integrated plasmids (Figs. 4A and B). The locations of the
binding sites for primers F and R used in these PCR
amplifications are indicated by the corresponding circled
letters in the diagrams of plasmid C in the Fig. 2 andplasmid D in Fig. 4B. The forward primer (primer F)
contains aClaI recognition sequence at its 30end so that it
will prime at the only segment in the genome containing a
ClaI site at a specific location downstream of a PGPEET .
The reverse primer (primer R) primes within the single
integrated copy of GFP in the genome. Examples of the
resultant PCR amplification products are shown in
Fig. 4A. All of the PCR products shown in Fig. 4A werecloned and sequenced to confirm their identity. Lanes 1, 2,
5, and 6 show the PCRamplification products of template
genomicDNAs from control cells. Lanes 1 and 2 show the
1.05-kb PCR product resulting from amplification of
template DNA from two cloned cell lines containing in-
tegrated plasmids B and C. In these cases, plasmid C does
not contain the [loxP(TERMGPEET )loxP] cassette and the
Fig. 4. (A) Photograph of an ethidium bromide-stained agarose gel containing the PCR products amplified with primers F and R (shown as circled
letters in Figs. 2 and 4B) from template genomic DNA isolated from procyclic cells containing the indicated plasmids (see Fig. 2) integrated into their
genome. White arrows point to the 2.2-kb and 1.08-kb PCR products generated with primers F and R. The genomic DNAs used as templates for the
PCR amplifications were obtained from independently derived T. brucei clones containing integrated plasmids B and C (lanes 1 and 2), plasmids B
and D (lanes 3 and 4), plasmids A and D (lane 5) or plasmids A and C (lane 6). (B) Diagrams depicting the sequences in the 2.2-kb PCR product
derived from the integrated plasmid D, shown in lanes 3 and 4 of Fig. 4A, and in the 1.08-kb PCR product derived from the deleted plasmid D, also
shown in lanes 3 and 4.
42 B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44
distance between the primer binding sites is about 1.05 kb.
In another control, lane 5 shows the 2.2-kb PCR ampli-fication product derived from genomic template DNA
of a cloned cell line containing integrated plasmids A
and D. In this case, plasmid D contains the [loxP(-
TERMGPEET )loxP] cassette but plasmid A does not en-
code CRE. Thus, excision of TERMGPEET should not
occur, and indeed sequence determination of the single
2.2-kb PCR product confirms that it contains the entire
[loxP(TERMGPEET )loxP] cassette. The control in lane 6shows the 1.05-kb PCR product resulting from amplifi-
cation of templateDNA froma cloned cell line containing
plasmids A and C. In this case, neither CRE nor the
[loxP(TERMGPEET )loxP] cassette is present, so a single
1.05-kb PCR product is expected and was found.
Lanes 3 and 4 show the results when the genomic
DNA templates for the PCR reaction are from two,
independently derived, cloned cell lines containingplasmids B and D that had been passaged in liquid
culture for about five weeks. Two PCR products occur,
one of 2.2 kb and the other of 1.08 kb, i.e., slightly larger
than the 1.05-kb fragment in the adjacent lanes 1 and 2.
Each of these PCR products was cloned and sequenced.
The 2.2-kb product was found to contain the same se-
quence as the single 2.2-kb PCR product in lane 5,
demonstrating it is derived from the intact [loxP(-TERMGPEET )loxP] cassette of the integrated plasmid D.
The 1.08-kb PCR product was found to possess the
same sequence as the 1.05-kb product derived from
plasmid C, except that it also contains one copy of loxP
(34-bp) between the ClaI and HindIII sites at which the
[loxP(TERMGPEET )loxP] cassette was originally in-
serted during the construction of plasmid D, as illus-
trated in Fig. 4B. Thus, the 1.08-kb PCR product isderived from genomic DNA in which TERMGPEET has
been excised, leaving a single loxP, which is consistent
with its excision by CRE recombinase. This result
demonstrates that the cell populations whose genomic
DNAs yielded the PCR products in lanes 3 and 4, andwhich were cloned populations initially, have become
two detectable populations with respect to their genome
after several weeks of passage in culture. One popula-
tion�s genomic DNA has retained the 2.2-kb region
bearing the [loxP(TERMGPEET )loxP] cassette, whereas
the other population�s genomic DNA has lost this cas-
sette to yield a 1.08-kb PCR product bearing a single
loxP at the original site of the cassette, despite the factthat CRE expression was not induced by tetracycline.
The cloned cell lines examined by PCR amplifications
(Fig. 4) were also analyzed for GFP expression by the
fluorescent activated cell sorter (FACS), as shown in
Fig. 5. Panels A, B, and C of Fig. 5 show the FACS
analyses of the same cell populations whose PCR anal-
yses are shown in lanes 3, 4, and 5 of Fig. 4A, respec-
tively. For example, lane 3 of Fig. 4A shows that aboutequal amounts of the 2.2- and 1.08-kb PCR products
were generated from the genomic DNA of this mixed
population. Correspondingly, panel A of Fig. 5 shows
that 49% of these same cells fluoresce green (open ar-
rowhead) and 51% do not (closed arrowhead), when the
trace is gated to resolve the two peaks. A comparison of
the PCR analysis in lane 4, Fig. 4A, and the FACS
analysis in panel B, Fig. 5, on the other independentlyderived mixed population of cells also yields consistent
results. The PCR analysis shows that the 1.08-kb PCR
product is more abundant than the 2.2-kb product,
whereas the gated FACS analysis reveals that 78% of the
cells fluoresce green and 22% do not. Similarly, lane 5,
Fig. 4A, contains a single 2.2-kb PCR product and panel
C, Fig. 5, shows that none of these cells fluoresce green.
The cells analyzed in lane 1, Fig. 4A, are the same cellsanalyzed in panel D, Fig. 5. In this case, all of the cells
should fluoresce green because they contain integrated
plasmid C, which does not have TERMGPEET between
Fig. 5. Analysis of GFP fluorescence in cloned procyclic T. brucei cells
containing the indicted plasmids integrated in their genome. One
million cells were collected, suspended in 1ml and their green fluo-
rescence determined by flow cytometry. The graphs are histograms of
relative GFP fluorescence plotted against frequency of events per
channel. The arrow with the closed arrowhead indicates the cell pop-
ulation that does not display green fluorescence and the arrow with the
open arrowhead indicates the cell population that fluoresces green. The
cells contained the indicated plasmids (see Fig. 2) integrated into their
genome. The cells analyzed in Panels A, B, and C are the same cells
analyzed by the PCR amplifications in lanes 3, 4, and 5 of Fig. 4A,
respectively. The cells analyzed in panel d are the same as those ana-
lyzed in lane 1 of Fig. 4A.
B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44 43
PGPEET and GFP. As described above, only one PCR
product of 1.05 kb is obtained from these cells (lane 1,Fig. 4A), consistent with the presence of integrated
plasmid C and the fact that all of these cells should be
green. However, FACS analysis reveals that only about
90% of the cells fluoresce green, a determination born
out by visual inspection of the cells in a fluorescence
light microscope. We do not know the reason for this
slight discrepancy, but the simplest interpretation is that
some of these cells have lost GFP while retaining BLE,which was used to select and maintain the cells trans-
fected with plasmid C.
4. Discussion
Only one transcription terminator sequence in T.
brucei has been reported to date (Berberof et al., 1996).This sequence of about 2 kb occurs immediately after a
5-kb cluster of four tightly packed protein-encoding
genes that are transcribed from PGPEET by an RNA
polymerase I-like activity (Berberof et al., 1996; Roditiand Clayton, 1999). This 2-kb terminator region was
originally found to contain at least three independent
elements capable of terminating transcription from
PGPEET . Each of these elements is, in turn, comprised of
multiple weaker elements. No obvious sequence simi-
larities exist in the three main elements. We chose the 30-distal of these three elements to use in the experiments
here, i.e., a 1.1-kb region that occurs between NruI andXhoI sites in the larger 2-kb sequence. We found that
when this 1.1-kb sequence is placed between PGPEET and
GFP, it does indeed prevent GFP expression, similar to
the original studies that showed when this region was
inserted between PGPEET and the gene for chloram-
phenicol acetyltransferase (CAT), it diminished CAT
transcription by 7–20-fold (Berberof et al., 1996). We
did not quantitate the extent to which it blocked GFP
expression from PGPEET in our system, but we did find
that it: (i) prevents the cells from appearing green visu-
ally in the fluorescent light microscope, and (ii) permits a
clear distinction to be made via FACS analysis between
cells that fluoresce green and those which do not because
of the presence of the terminator (Fig. 5).
In addition to the experiments described here, several
other experiments were conducted whose results areconsistent with those described. First, a plasmid identical
to plasmid D (Fig. 2) was constructed, except
TERMGPEET was not flanked by loxP elements. When
this plasmid was inserted into the genome of cells bearing
plasmid B, no evidence was obtained for the possible ex-
cision of TERMGPEET by CRE, i.e., the permanently
transfected cells did not fluoresce green under any con-
ditions nor did green cells appear with time. This resultindicates that the flanking loxP elements are required for
excision of the terminator sequence by CRE. Secondly, in
another set of experiments plasmid D was inserted into
the T. brucei 29–13 genome lacking plasmid B, and then
the cells were transiently transfectedwith circular plasmid
B without selection for plasmid B�s antibiotic resistance
gene (BLEO). Transiently transfected plasmids can enter
as many as 80% of the cells during electroporation (La-Count et al., 2000), but they do not replicate in the cells as
a circular plasmid and are unlikely to integrate into the
genome, so they are lost as the cells divide. None of the
cells bearing plasmidDdisplayed green fluorescence prior
to the transient transfection with plasmid B. Two days
after the transient transfection, however, an occasion
green cell (�1 in 500) was observed visually in the fluo-
rescent microscope. No green cells were observed whenplasmid A was the transiently transfected plasmid. In the
plasmid B case, the few green cells persisted as the culture
was passaged but their percentage did not increase with
time, suggesting that transient expression of CRE from
transiently transfected plasmid B caused the excision of
the loxP-flanked TERMGPEET in these few cells. It should
be noted that this result occurred without tetracycline
44 B. Barrett et al. / Experimental Parasitology 106 (2004) 37–44
induction, again consistent with the interpretation that:(i) low-level expression of CRE occurs from plasmid B in
the absence of tetracycline, and (ii) this low-level expres-
sion is sufficient to excise the loxP-flanked TERMGPEET .
This result indicates that a short-term, low-level expres-
sion of CRE is sufficient to trigger a recombination event
at loxP sites.
Thus, the experiments summarized here demonstrate
that CRE recognizes the exogenously added 34-bp loxP
sequence in the T. brucei genome and can conduct pre-
cise excision of a DNA region flanked by directly ori-
ented loxP elements. From a practical standpoint,
however, it appears that the presence of too much CRE
is deleterious to the T. brucei cells, killing them when
CRE�s expression is induced by tetracycline from
PGPEET on a pLEW100-based plasmid (plasmid B in
Fig. 2). Since these T. brucei experiments were con-cluded, high-level expression of CRE has also been
shown to be highly toxic to mammalian and Drosophila
cells, due apparently to pseudo-loxP sites in the genome
that can serve substrates for CRE and lead to severe
chromosomal damage (Andreas et al., 2002; Heidmann
and Lehner, 2001; Loonstra et al., 2001). It is likely that
the same toxic recombination events occur in T. brucei.
With these recentmammalian andDrosophila results inmind, it is clear that for the CRE-loxP system to be useful
for analysis of gene function and regulation in T. brucei,
either: (i) a ‘‘low-level’’ expression system for CRE must
be found in which CRE expression can be completely
turned off until transiently required, and/or (ii) amodified
form of CRE is identified that has much less recombina-
tion activity on pseudo-loxP sites. Experimental muta-
tions of the PGPEET in pLEW100 to reduce its efficiency orsimilar mutations of the CRE coding sequence to reduce
its activity may be necessary to achieve this goal. Alter-
natively, another site-specific recombination system
might be used, such as the yeast flipase (FLP) system in
which the FLP recombinase has recently been shown to
have only 10% of the recombination activity at its target
sequence compared toCREat loxP (Andreas et al., 2002).
The observation that during five weeks of culturedgrowth, 22–50% of the cells containing CRE underwent
CRE-mediated excision at the loxP sites (Figs. 4 and 5),
even though CRE expression was repressed by two adja-
cent Otet elements, demonstrates that only small amounts
of a recombinase are needed to catalyze site-specific re-
combination in T. brucei.
Acknowledgments
We thank Dr. Justin Fishbaugh of the Flow Cy-
tometry Facility at the University of Iowa for expert
advice and technical assistance. This work was sup-
ported in part by National Institutes of Health (NIH)
Grants AI10512 and AI40591.
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