RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-612
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Last modified: 9 March 2011, 21:16
*RMgm-612| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene disruption |
| Number of attempts to introduce the genetic modification | 4 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 21288823 |
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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 | Not applicable |
| Other information parent line | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | A. Ecker; V. Lakshmanan, D.A. Fidock |
| Name Group/Department | Department of Microbiology and Immunology |
| Name Institute | Columbia University Medical Center |
| City | New York |
| Country | USA |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1219500 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0709000 | ||||||||||||||||||||||||
| Gene product | chloroquine resistance transporter | ||||||||||||||||||||||||
| Gene product: Alternative name | CRT | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | Plasmid double cross-over | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||
| Restriction sites to linearize plasmid | HpaI, ScaI | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The unsuccessful attempts to select for mutants with a disrupted/deleted crt gene, indicates an essential role of CRT during asexual blood stage development (see also below). In earlier studies in P. falciparum, the failure to disrupt pfcrt (MAL7P1.27) suggested an essential role of CRT for asexual blood stage viability (Waller et al., 2003, J. Biol. Chem. 278, 33593-601). See RMgm-610 and RMgm-611 for P. berghei mutants in which the crt gene of P. berghei is replaced by the P. falciparum crt gene (MAL7P1.27) of the chloroquine sensitive HB3-strain and chloroquine resistant 7G8-strain, respectively. The analyses of these mutants demonstrate that CRT of P. falciparum can complement the function of CRT of P. berghei. The knockout/disruption plasmid was constructed in an identical manner to the allelic replacement plasmids described in RMgm-610 and RMgm-611, except that it lacks the pfcrt coding sequence. PCR analysis detected a minor subpopulation of pbcrt-deleted parasites in 2 of 4 independent transfection experiments, with the majority of the parasites maintaining the knockout plasmid episomally (data not shown). However, the knockout population could not be cloned, indicating that pbcrt is likely required for 'productive' asexual blood stage development. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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