RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-618
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Last modified: 21 December 2011, 15:56
*RMgm-618| 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 | 3 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 21651909 |
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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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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | A.C. Raabe; O. Billker; K. Wengelnik |
| Name Group/Department | UMR5235, CNRS |
| Name Institute | Université Montpellier 2 |
| City | Montpellier |
| Country | France |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1211900 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1013500 | ||||||||||||||||||||||||
| Gene product | phosphoinositide-specific phospholipase C | ||||||||||||||||||||||||
| Gene product: Alternative name | PI-PLC | ||||||||||||||||||||||||
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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 | |||||||||||||||||||||||||
| 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 | Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of malaria parasites. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. The Plasmodium protein contains all domains typically found in PI-PLCs of the delta class but is almost twice as long as their orthologues in mammals. The negative attempts to disrupt the gene indicates an essential role of PI-PLC during asexual blood stage growth and multiplication. In addition to attempts to disrupt the Pbplc locus by double cross-over homologius recombination, the Pbplc locus was targeted by single homologous recombination in the beginning of the plc open reading frame. The hdhfr resistance cassette would in this way be inserted into the plc coding sequence after amino acid 368, upstream of the X and Y catalytic domains. However, in three independent experiments this construct did not yield viable knock-out parasites either indicating that either PI-PLC was essential during blood stage development or the locus was refractory to recombination. A promoter exchange strategy was chosen that would prove that the plc locus could be targeted by genetic modification and, at the same time, might allow modification of the plc expression level. The targeting vector was designed based on the single cross-over knock-out construct described above. By placing an alternative promoter in front of the homology region needed for recombination, integration of the construct places a new promoter in front the plc coding sequence. The expression profile of the cGMP-dependent protein kinase gene (pkg; PF14_0346; PBANKA_100820) resembles the one of plc in P. falciparum. The pkg promoter was therefore used for a ‘proof of principle’ approach. Upon transfection pyrimethamine resistant parasites were obtained and were cloned by limiting dilution. PCR analysis indicated that the construct had integrated correctly into the genome, thereby demonstrating that the plc locus was generally available for recombination and that the promoter replacement itself was possible. Thus, the inability to obtain plc knock-out parasites is most likely due the fact that PI-PLC activity is essential during asexual erythrocytic development. However, in an attempt to over-express the plc gene by using the strong promoter of the elongation factor-1alpha gene (ef1a) no parasites were obtained with correct integration in three independent transfections, suggesting that over-expression of plc was not supported by the parasite. In addition, several other promoter exchange constructs also did not integrate into the genome (data not shown) indicating that plc expression might me tightly regulated by the parasite Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565). The gene is likely essential for asexual proliferation as integration of the KO vector was not achieved. Accesibility of the locus to recombination was verified by C-terminal tagging. | ||||||||||||||||||||||||
| 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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