6 research outputs found
Comparison of growth behavior of YM and <i>PYΔpy01365.</i>
<p>A- Parasitaemia of BALB/c mice infected with 10<sup>4</sup> parasites on
day 0 was taken daily. The average parasitaemia of 5 mice for both YM
and <i>PYΔpy01365</i> is represented. Error bars are given
for each time point. <b>†</b> Indicates death of animals.
B- Average Selective index of 5 BALB/c mice infected with either YM or
<i>PYΔpy01365.</i> Parasites smears were analyzed when
parasitaemia was in the range of 5–15%. Differences in SI
between YM and <i>PYΔpy01365</i>were significant
(p<0.01).</p
Differences in expression of Py235 recognized by 25.77 in YM or <i>PYΔpy0136</i> parasites.
<p>A- Immunofluorescence Assays of YM and
<i>PYΔpy01365(NF1</i>) and
<i>PYΔpy01365(NF2</i>) with the protective monoclonal
antibody 25.77 (Py235). Fewer schizonts in
<i>PYΔpy01365</i> reacted specifically with the Py235
specific antibody (circled). The specific antibodies that reacted with
the schizonts were detected with Alexa Fluor labeled anti-mouse IgG. The
fluorescent images (individual stains and merged) and the bright-field
are shown. B- Immunofluorescence Assays of YM and
<i>PYΔpy01365</i> parasites with mcAb 25.77 (Py235)
and a rabbit serum against the rhoptry protein MAEBL. The specific
antibodies that reacted with the schizonts were detected with Alexa
Fluor labeled goat anti rabbit (or anti-mouse) IgG. The fluorescent
images (individual stains and merged) and the bright-field are shown. C-
Quantification of the number of schizonts that are MAEBL and Py235
positive. YM and <i>PYΔpy01365</i> parasites were stained
with mcAb 25.77 and a rabbit serum against the rhoptry protein MAEBL. A
total of 200 MAEBL positive schizonts were counted and their mcAb 25.77
staining was determined. Comparison of double labeled parasites showed a
significant difference between YM and <i>PYΔpy01365</i>
parasites (p< 0.01).</p
Disruption of <i>py01365</i> using homologous recombination.
<p>A- genomic locus MALPY00360 coding for <i>py01365</i> showing
the two regions (blue and red) used for targeting this locus by a double
cross-over strategy. Homologous recombination with the linearized
plasmid containing the selectable marker flanked by the targeting
sequences results in the Py01365 KO locus. Restriction sites used for
Southern blot analysis as well as the location of the primer pairs A-F,
A-R and B-F, B-R important for PCR screening of both the 5′ and
3′ integration event as well as region used for Southern blot
probe are also indicated. B- PCR screening of 5′ (A) and 3′
(B) integration events in both wild type (YM) and knock out (KO)
parasites using primers A-F, A-R and B-F, B-R respectively. Both primer
pairs are only expected to give a product if integration has occurred.
C- Southern blot screening of parasites for correct integration. (1)
BstBI digested DNA obtained from wild type (YM) as well as transfected
parasites (K1, K2 and K3) and the transfection plasmid (Pl) was analyzed
by Southern blot using a <i>PY01365</i> specific probe (region
indicated in red). The expected fragment of ∼7.4 kb can be seen in
all three transfected parasite lines. (2) Transfected clone K1 and K3
were subsequently cloned out by limiting dilution and again screened by
Southern blot. Single parasite clone K1-C1 and K3-C2 were selected for
further analysis, and were renamed
<i>PYΔpy01365(NF1</i>)and
<i>PYΔpy01365(NF2</i>), respectively.</p
Unique PCR primers used for Real Time-PCR.
<p>Unique PCR primers used for Real Time-PCR.</p
Erythrocyte binding assay of parasite culture supernatant from both YM and <i>PYΔpy01365</i>.
<p>Western blot analysis using mcAb 25.77 of equal amounts of parasite
culture supernatant as well as proteins bound to erythrocytes from A) YM
and <i>PYΔpy01365(NF2</i>) as well as B) YM and
<i>PYΔpy01365(NF1</i>).</p
Transcription of <i>py235</i> in YM and <i>PYΔpy01365</i> parasites.
<p>Analysis of changes of the transcription pattern of different
<i>py235</i> members by quantitative reverse transcription
- real time-PCR. Analysis of transcription levels of 11 different
<i>py235</i> members in YM (red) and
<i>PYΔpy01365(NF1</i>) (blue) and
<i>PYΔpy01365(NF2</i>) (green). Results are expressed
as percent of total <i>py235</i> transcription. *
indicates statistically significant differences in the transcription
levels of a gene between YM and <i>PYΔpy01365</i>
parasites (p<0.05).</p