CD4+ and CD8+ T cells from the harvested spleens of the
vaccinated and the unvaccinated mice is performed by using
the Mouse Pan T Cell Isolation Kit and MS columns (Miltenyi
Biotec). The cells from the same group are pooled on the same
day, and ~10
6.3 T cells/mouse are administered via the retroorbital route to a new set of naive female C57BL/6 mice. In a
similar fashion, sera from either the vaccinated or unvaccinated
groups and matching groups are collected and pooled together.
100 μL/mouse of the pooled serum is injected retro-orbitally
to a new set of naive female C57BL/6 mice. After 1 day of
adoptive transfer, the mice in all groups are challenged with
2 MLD 50 of WSN or 2 MLD 50 of HK68/H3 intranasally.
3.7 Genetic
Modifications
in Parasites
1. Disruption of the p36p to create p36pÀ/À Parasite Lines: A
p36p replacement vector containing pyrimethamine-resistant
Toxoplasma gondii (tg) dhfr/ts and dhfr selectable marker is
introduced in vector b3D.D T . H. D b
>
>
in order to disrupt the
p36p locus (see Note 7). The vector also comprises gfp placed
under the control of the constitutive pbef-1aa promoter [18–
20] and a fragment of 2 kb of the D-type small subunit (dssu)
rRNA gene of P. berghei [21]. The linearized vector has the
propensity to integrate in C-type small subunit (cssu) and/or
dssu. Integration into cssu cause no change to the parasitic
phenotype [45]. Wild-type (WT) P. berghei ANKA strains
(clone 15cy1) are used to generate p36p
À parasites. KO1
p36p
À parasites are cotransfected with the gfp vector to create
gfp-expressing p36p
À mutants [46]. Transformed parasites are
selected by treating infected animals with WR99210 (16 mg/
kg bodyweight) [20]. KOGFP clone containing gfp at the site
of cssu is selected. Confirmation of correctly integrated constructs is analyzed by RT-PCR and Southern analysis
[46]. PCR amplification of WT and p36p
À parasites is performed by using primers specific for the WT ((L1362) (see
Subheading 2.7.1) and L1363 (see Subheading 2.7.1)) and
disrupted ((L1389) (see Subheading 2.7.1) and L313 (see Subheading 2.7.1)). WT and p36p
À
:gfp parasites are amplified by
using primers specific for WT ((L270) (see Subheading 2.7.1)
and L271 (see Subheading 2.7.1)) and disrupted cssu ((L270
and L635 (see Subheading 2.7.1)). Primers (1389 and 313)
produced the fragment of 1.0 kb in KOGFP parasites with
disrupted p36p locus. RT-PCR is performed on WT and
p36p
À sporozoites RNA as described by Invitrogen. In order
to amplify cDNA derived from the p36p
À or circumsporozoite
(CS) gene, primers (L1425 (see Subheading 2.7.1) and L1363
(see Subheading 2.7.1)), and (L1502 (see Subheading 2.7.1)
and L1503 (see Subheading 2.7.1)) are used.
350
Dipasree Hajra et al.
vaccinated and the unvaccinated mice is performed by using
the Mouse Pan T Cell Isolation Kit and MS columns (Miltenyi
Biotec). The cells from the same group are pooled on the same
day, and ~10
6.3 T cells/mouse are administered via the retroorbital route to a new set of naive female C57BL/6 mice. In a
similar fashion, sera from either the vaccinated or unvaccinated
groups and matching groups are collected and pooled together.
100 μL/mouse of the pooled serum is injected retro-orbitally
to a new set of naive female C57BL/6 mice. After 1 day of
adoptive transfer, the mice in all groups are challenged with
2 MLD 50 of WSN or 2 MLD 50 of HK68/H3 intranasally.
3.7 Genetic
Modifications
in Parasites
1. Disruption of the p36p to create p36pÀ/À Parasite Lines: A
p36p replacement vector containing pyrimethamine-resistant
Toxoplasma gondii (tg) dhfr/ts and dhfr selectable marker is
introduced in vector b3D.D T . H. D b
>
>
in order to disrupt the
p36p locus (see Note 7). The vector also comprises gfp placed
under the control of the constitutive pbef-1aa promoter [18–
20] and a fragment of 2 kb of the D-type small subunit (dssu)
rRNA gene of P. berghei [21]. The linearized vector has the
propensity to integrate in C-type small subunit (cssu) and/or
dssu. Integration into cssu cause no change to the parasitic
phenotype [45]. Wild-type (WT) P. berghei ANKA strains
(clone 15cy1) are used to generate p36p
À parasites. KO1
p36p
À parasites are cotransfected with the gfp vector to create
gfp-expressing p36p
À mutants [46]. Transformed parasites are
selected by treating infected animals with WR99210 (16 mg/
kg bodyweight) [20]. KOGFP clone containing gfp at the site
of cssu is selected. Confirmation of correctly integrated constructs is analyzed by RT-PCR and Southern analysis
[46]. PCR amplification of WT and p36p
À parasites is performed by using primers specific for the WT ((L1362) (see
Subheading 2.7.1) and L1363 (see Subheading 2.7.1)) and
disrupted ((L1389) (see Subheading 2.7.1) and L313 (see Subheading 2.7.1)). WT and p36p
À
:gfp parasites are amplified by
using primers specific for WT ((L270) (see Subheading 2.7.1)
and L271 (see Subheading 2.7.1)) and disrupted cssu ((L270
and L635 (see Subheading 2.7.1)). Primers (1389 and 313)
produced the fragment of 1.0 kb in KOGFP parasites with
disrupted p36p locus. RT-PCR is performed on WT and
p36p
À sporozoites RNA as described by Invitrogen. In order
to amplify cDNA derived from the p36p
À or circumsporozoite
(CS) gene, primers (L1425 (see Subheading 2.7.1) and L1363
(see Subheading 2.7.1)), and (L1502 (see Subheading 2.7.1)
and L1503 (see Subheading 2.7.1)) are used.
350
Dipasree Hajra et al.