2. Neonatal Mice: BALB/c mice (15 day old) (Vital River Beijing) of weight 6–9 g.
3. Ferrets: Healthy young adult outbred female ferrets (Mustela
putorius furo; between 4 and 5 months of age) (purchased
from a commercial breeder (Wuxi)).
2.6.6 Mouse
Immunization
and Challenge
1. Mouse Pan T Cell Isolation Kit and MS columns (Miltenyi
Biotec).
2.6.7 In Vivo Challenge
Using HPAI Virus H5N1
1. Female BALB/c mice (Mus musculus) aging of 6–8 weeks
(purchased from Charles River Laboratories).
2. Microisolator cages with ventilation facility having negative
pressure with HEPA-filtered air and a 12:12 h light–dark cycle.
3. BSL3 facilities at the Pasteur Institute of Cambodia.
4. Pentobarbital sodium (75 mg/kg; Sigma).
2.7 Genetic
Modifications
in Parasites [17]
2.7.1 Generation
of the p36p
À Parasite Lines
1. Vector harboring the pyrimethamine-resistant Toxoplasma gondii (tg) dhfr/ts gene, human (h) dhfr selectable marker, and gfp
placed under the control of the constitutive pbef-1aa promoter
[18–20] and a 2-kb fragment of the D-type small subunit (dssu)
rRNA gene of Plasmodium berghei [21].
2. P. berghei wild type (clone 15cy1; ANKA strain).
3. WR99210 (16 mg/kg bodyweight) [20].
4. Primer Pairs Used.
(a) WT L1362 5
0 -CCGCTCGAGACCTTAGGACACTTTG
AAATTTG-3
0 .
(b) L1363 5
0 -CCGCTCGAGCTACTCATAATAAGAAGAA
GAGGTAC-3
0 .
(c) Disrupted L1389 5
0 -ATTTTGCAACAATTTTATTCTT
GG-3
0 .
(d) L313 5
0 -ACGCATTATATGAGTTCATTTTAC-3
0 .
(e) WT L270 5
0 -GTGTAGTAACATCAGTTATTGTGTG3
0 .
(f) L271 5
0 -CTTAGTGTTTTGTATTAATGACGATTTG-3
0 .
(g) Disrupted cssu L270 and L635 5
0 -TTTCCCAGTCACG
ACGTTG-3
0 .
5. RNA Isolation and RT-PCR (Invitrogen).
6. Primers for amplification of cDNA derived from the p36p
À or
circumsporozoite (CS) gene.
(a) L1425 (5
0 -GAAATGAATATGTCGGTACTATG-3
0 ).
(b) L1363
(5
0 -CCGCTCGAGCTACTCATAATAAGAAG
AAGAGGTAC-3
0 ).
Live Vaccine Attenuation
339
3. Ferrets: Healthy young adult outbred female ferrets (Mustela
putorius furo; between 4 and 5 months of age) (purchased
from a commercial breeder (Wuxi)).
2.6.6 Mouse
Immunization
and Challenge
1. Mouse Pan T Cell Isolation Kit and MS columns (Miltenyi
Biotec).
2.6.7 In Vivo Challenge
Using HPAI Virus H5N1
1. Female BALB/c mice (Mus musculus) aging of 6–8 weeks
(purchased from Charles River Laboratories).
2. Microisolator cages with ventilation facility having negative
pressure with HEPA-filtered air and a 12:12 h light–dark cycle.
3. BSL3 facilities at the Pasteur Institute of Cambodia.
4. Pentobarbital sodium (75 mg/kg; Sigma).
2.7 Genetic
Modifications
in Parasites [17]
2.7.1 Generation
of the p36p
À Parasite Lines
1. Vector harboring the pyrimethamine-resistant Toxoplasma gondii (tg) dhfr/ts gene, human (h) dhfr selectable marker, and gfp
placed under the control of the constitutive pbef-1aa promoter
[18–20] and a 2-kb fragment of the D-type small subunit (dssu)
rRNA gene of Plasmodium berghei [21].
2. P. berghei wild type (clone 15cy1; ANKA strain).
3. WR99210 (16 mg/kg bodyweight) [20].
4. Primer Pairs Used.
(a) WT L1362 5
0 -CCGCTCGAGACCTTAGGACACTTTG
AAATTTG-3
0 .
(b) L1363 5
0 -CCGCTCGAGCTACTCATAATAAGAAGAA
GAGGTAC-3
0 .
(c) Disrupted L1389 5
0 -ATTTTGCAACAATTTTATTCTT
GG-3
0 .
(d) L313 5
0 -ACGCATTATATGAGTTCATTTTAC-3
0 .
(e) WT L270 5
0 -GTGTAGTAACATCAGTTATTGTGTG3
0 .
(f) L271 5
0 -CTTAGTGTTTTGTATTAATGACGATTTG-3
0 .
(g) Disrupted cssu L270 and L635 5
0 -TTTCCCAGTCACG
ACGTTG-3
0 .
5. RNA Isolation and RT-PCR (Invitrogen).
6. Primers for amplification of cDNA derived from the p36p
À or
circumsporozoite (CS) gene.
(a) L1425 (5
0 -GAAATGAATATGTCGGTACTATG-3
0 ).
(b) L1363
(5
0 -CCGCTCGAGCTACTCATAATAAGAAG
AAGAGGTAC-3
0 ).
Live Vaccine Attenuation
339