SummaryRMgm-1462
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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 | Unknown |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27316546 |
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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 | Ebine K; Saito-Nakano Y |
Name Group/Department | Department of Parasitology |
Name Institute | National Institute of Infectious Diseases |
City | Tokyo |
Country | Japan |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1409100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1310600 | ||||||||||||||||||||||||||
Gene product | secretory complex protein 61 alpha | ras-related protein Rab-5B | ||||||||||||||||||||||||||
Gene product: Alternative name | RAB5b; Sec61-alpha; Rab GTPase 5b | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The P. berghei Rab5b gene was attempted to replace with Toxoplasma gondii Rab5b (TGME49_207460) | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) 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 | |||||||||||||||||||||||||||
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 | pL0006 was obtained from MR4 and was used to make a panel of transfection constructs. To replace the genomic sequence encoding PbRab5b, 500 bp upstream and downstream of the PbRab5b locus were cloned into HindIII–PstI and XhoI–EcoRI sites of pL0006, respectively (PbRab5b-KO plasmid). A series of open reading frames (ORFs) comprising the sequences encoding PbRab5b, mAG (monomeric Azami Green), and the terminator region of P. berghei dihydrofolate reductase (PbDT) were PCR-amplified with overlapping oligonucleotides and were then inserted into the PstI site of the PbRab5b-KO plasmid using an In Fusion HD cloning kit (Clontech, USA) to yield PbRab5b-mAG plasmid. For complementation analysis, each gene-of-interest (GOI) was PCR-amplified, and an In Fusion HD cloning kit was used to replace the PbRab5b ORF in the PbRab5b-mAG plasmid. In this study the endogenous PbRab5b locus was replaced with the following Rab5 homologues: PbRab5b fused to monomeric Azami Green (RMgm-1460); Rab5b from P. falciparum (PfRab5b, PF3D7_1310600)(RMgm-1461), It was also attempted to replace it with Rab5b from the distantly-related apicomplexan parasite Toxoplasma gondii (TgRab5b, TGME49_207460)(RMgm-1462), and the other conventional Rab5 isoforms of P. berghei, PbRab5a, PBANKA_030800 (RMgm-1463) and PbRab5c PBANKA_020650 (RMgm-1464). Replacement with PfRab5b was successful; Replacement with TgRab5b, PbRab5a and PbRab5c was not successful. First, the genomic locus encoding PbRab5b was replaced with a fragment encoding PbRab5b fused to monomeric Azami Green (mAG). This transgenic parasite grew as well as the wild type, indicating that the PbRab5b-mAG fusion protein was functional. The PbRab5b genomic locus was also successfully replaced with PfRab5b fused to mAG (PfRab5b-mAG), indicating that PfRab5b complemented the function of PbRab5b. This result is supported by the presence of a completely conserved effector sequence (HQVTIGAAFL) between PbRab5b [amino acid residues (aa) 60–69] and PfRab5b (aa 63–72) that specifies the function of Rab proteins. In contrast, P. berghei-transfected with TgRab5b-mAG, which has a similar effector sequence (aa 66–75, HEVTIGAAFL, with the underline indicating different residue) in addition to a characteristic insertion sequence (aa 165–178), did not functionally complement PbRab5b. These results suggest that the molecular function of Rab5b in P. berghei is conserved with P. falciparum, but not with Toxoplasma gondii. After drug selection, parasites transfected with plasmids replacing PbRab5b with conventional Rab5 isoforms, PbRab5a and PbRab5c, were not obtained, suggesting Rab5a and Rab5c were unable to complement endogenous PbRab5b. To identify the functional regions of PbRab5b, a panel of chimeric constructs, which consisted of PbRab5b with replacements of equivalent regions of PbRab5a or PbRab5c, was expressed. The following chimeric constructs did not complement endogenous PbRab5b; aa 1–34, 1–69, or 1–92 in PbRab5b-mAG proteins were replaced with equivalent regions from PbRab5a, PbRab5b–5a #1, #2, and #3, respectively), and aa 1–69 in PbRab5b-mAG was replaced with corresponding sites of PbRab5c. The only chimera that complemented the PbRab5b locus was a construct that included the entire GTPase motif of Rab5b (aa 1–192, PbRab5b–5a #4). These results indicate that PbRab5b functions differently from PbRab5a or PbRab5c, and that the GTPase activity is required for a proper Rab5b function. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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