RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-363

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1218100; Gene model (P.falciparum): PF3D7_0320400; Gene product: oocyst capsule protein Cap380, putative (PbCap380, Plasmodium oocyst capsule protein)
Phenotype	Oocyst; Sporozoite;

Last modified: 29 December 2009, 15:02

*RMgm-363

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 18248630
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/Researcher	P. Srinivasan; M. Jacobs-Lorena
Name Group/Department	Malaria Research Institute, Department of Molecular Microbiology and Immunology
Name Institute	Johns Hopkins School of Public Health
City	Baltimore
Country	US
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Name of the mutant parasite
RMgm number	RMgm-363
Principal name	PbCap380(−)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Not different from wild type
Oocyst	Normal numbers of midgut ookinetes are produced. Ookinetes can invade and traverse the midgut epithelium comparable to wild type ookinetes. Ookinetes transform efficiently in oocysts but the number of oocysts declined as development proceeded. When analysed at day 15 after the infectious blood meal, neither mature oocysts nor sporozoites could be detected. In a few mosquitoes small, disintegrating oocysts were observed.
Sporozoite	No mature oocysts and (oocyst-derived) sporozoites are formed
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of PbCap380 (Plasmodium oocyst capsule protein). Protein (function) Anti-PbCap380 antibody staining showed that PbCAPp380 can be detected soon after the ookinete transforms into an oocyst (36 h) and remains on the oocyst periphery till sporozoites begin to be released at around day 15. Confocal microscopy of day 15 oocysts double-labelled with anti-PbCap380 and anti-PbCSP antibodies show that PbCap380 localizes externally to the CS protein, suggesting that it is part of the protective capsule. Immuno-electron microscopy confirmed that PbCap380 localizes to the capsule. Phenotype The phenotype analyses indicate an essential role of PbCap380 for development of the oocyst and its involvement in the formation of the oocyst capsule (wall). Mutant parasites form oocysts in normal numbers but are gradually eliminated. Additional information Quantitative RT-PCR analysis showed that PbCap380 is expressed only during oocyst differentiation in the mosquito. Two gene models exist: PB000071.00.0 and PB300510.00.0 Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1218100

Gene Model P. falciparum ortholog

PF3D7_0320400

Gene product

oocyst capsule protein Cap380, putative

Gene product: Alternative name

PbCap380, Plasmodium oocyst capsule protein

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

Plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Partial

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

pbdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The mutant has been generated using a construct that disrupt the gene by single cross-over integration (insertion vector). The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.

Additional remarks selection procedure

Primer information: Primers used for amplification of the target sequences Click to view information

Primer information: Primers used for amplification of the target sequences Click to hide information

Sequence Primer 1	5'-GCATGTAAAGTAATAAAACCATCTACA-3'
Additional information primer 1	IntgF
Sequence Primer 2	5'-AGGTGTAAATAATGATATGAAACCT-3'
Additional information primer 2	IntgR
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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