RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-741
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Last modified: 28 April 2012, 22:40
*RMgm-741| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene mutation |
| Number of attempts to introduce the genetic modification | 4 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 22355110 |
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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 | M. Zhang; V. Nussenzweig |
| Name Group/Department | Department of Pathology |
| Name Institute | New York University School of Medicine |
| City | New York |
| Country | USA |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0212100 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0728000 | ||||||||||||||||||||||||||
| Gene product | eukaryotic translation initiation factor 2 subunit alpha, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | eIF2α | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | WT eIF2α replaced by mutated eIF2α in which ser59 was substituted with aspartic acid | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | 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 | XhoI, AscI | ||||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | Because the function of PK4 is to phosphorylate ser59 of Plasmodium eIF2α, the importance of eIF2α for parasite development in erythrocytes was tested. For this, the WT eIF2α in P. berghei was replaced by alleles encoding eIF2α in which ser59 was substituted with nonphosphorylatable alanine (RMgm-740), or with threonine, which is structurally similar to serine, or with aspartic acid (described here),which mimics a phosphorylated serine, using replacement plasmids pBCeIF2α_S/* (A, T, D). As a control, plasmid pBCeIF2α_S/S was used which contains a synonymous mutation in the ser59 codon (TCA to TCg) tagged by a unique BstBI restriction site, for evaluating the frequency of mutation correction during double cross-over recombination of the plasmid series. The plasmids were independently transfected in P. berghei blood-stage parasites, and then the recombinant parasites were selected under pyrimethamine pressure in mice and analyzed by PCR analysis of the eIF2α gene using primers that amplify a 1,280-bp eIF2α fragment encompassing the ser59 codon from gDNA. In three independent experiments analyzing populations obtained after transfection of the control plasmid pBCeIF2α_S/S (S/S populations), the majority of the PCR fragments contained the BstBI-tagged S/S mutation. Therefore, a mutation in ser59 codon in the plasmid was frequently transferred to the endogenous eIF2α during plasmid recombination as long as it did not affect eIF2α function. Likewise, eIF2α S/T mutant parasites were obtained in three independent transfections of plasmid pBCeIF2α_S/T, indicating that threonine functionally replaced serine at position 59 in eIF2α. The S/S and S/T gametocytes were able to infect mosquitoes and generated normal numbers of sporozoites. The salivary gland sporozoites normally infected mice after a mosquito bite or i.v. injection. In contrast, when plasmid pBCeIF2α_S/A was transfected into WT P. berghei, only one resistant population was obtained in four independent transfection experiments. However, PCR analysis of the resistant parasite population showed that the MscI-tagged S/A mutation had been corrected during plasmid integration. In agreement with this finding, parasite clones from the resistant population contained only the recombinant eIF2α locus lacking the S/A mutation. We conclude that the eIF2α S/A mutation is deleterious to parasite viability. Similarly, transfection of plasmid pBCeIF2α_S/D into WT P. berghei failed to generate any resistant parasite population in three independent experiments. Presumably, the eIF2α S/D mutation inhibited protein synthesis, but this inhibition could not be reversed as normally occurs by removal of PO4 from eIF2α-P, which is required for the continuation of the life cycle. We cannot exclude, however, that the S/D mutation inactivates the central function of eIF2α in translation. In conclusion, the parasite blood stages bearing eIF2α S/A or S/D mutations were not viable, whereas the S/S or S/T mutant parasites were able to complete their life cycles successfully. The site-directed mutagenesis of PbeIF2α Ser59 was engineered by using the overlap extension PCR method. To generate the allelic replacement construct for PbeIF2α Ser59 substitutions, two fragments (−1,500 bp to −1,000 bp and −1,000 bp to +1,000 bp with mutations) were amplified and inserted on opposing ends of the hDHFR cassette of the pBC_hDHFR vector. The hDHFR cassette is under the control of the 5′UTR of EF1α and 3′ UTR of dihydrofolate reductase-thymidylate synthase (DHFR-TS). The allelic replacement vectors pBCeIF2α_S/* (A, T, D, or S) were linearized with XhoI and AscI restriction endonucleases, transfected into P. berghei ANKA merozoites, and then selected and single-cloned in mice. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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