SummaryRMgm-5218
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35649358 |
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 507cl1 (RMgm-7) |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Lahree A, Mota MM |
Name Group/Department | Instituto de Medicina Molecular- João Lobo Antunes (iMM-JLA), Faculdade de Medicina |
Name Institute | Universidade de Lisboa |
City | Lisboa |
Country | Portugal |
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Name of the mutant parasite | |
RMgm number | RMgm-5218 |
Principal name | PbRab5b_HA |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | The cellular localization of P. berghei Rab5b during the liver stage was investigated using parasite lines expressing this protein with a C-terminal monomeric Azami-Green (PbRab5b_mAG; 1460) tag or 2x C-terminal hemagglutinin (HA)-tag (PbRab5b_HA). The majority of the PbRab5b signal was in the parasite cytosol with a small peripheral fraction detected in close proximity to the PVM and in the tubovesicular network (TVN). Mature EEFs presented a higher peripheral PbRab5b signal, and the overall signal also increased with EEF development. |
Additional remarks phenotype | Mutant/mutation Evidence is presented for the following: Other mutants |
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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 | ras-related protein Rab-5B | ||||||||||||||||||||||||||
Gene product: Alternative name | RAB5b; secretory complex protein 61 alpha; Sec61-alpha; Rab GTPase 5b | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | HA | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
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 | For the generation of HA-tagged Rab5b expressing P.berghei parasite line, a double homologous recombination strategy was employed. The P.berghei rab5b (Pbrab5b) gene was amplified with a high fidelity polymerase (Phusion polymerase, Thermo Scientific) using the primers 1 and 2 to insert the 2x-HA tag on the Pbrab5b amplicon. 3′ÚTR of Pbrab5b gene was amplified using the primers 3 and 4. Blunt-ended PCR products were purified with QIAquick PCR purification kit (Qiagen, Hilden, Germany) and used for insertion in pJet-1.2 cloning vector (CloneJET PCR cloning kit) according to manufacturer's instructions. The ligation mixture was used for bacterial transformation of E. coli DH5α chemically competent cells (produced in-house according to the manufacturer's protocol, NZYCompetent Cells Preparation Buffer, NZYtech). Transformed cells were spread on LB-Ampicillin (100 μg/mL, Sigma) agar and incubated at 37°C for 16–24 h, for selection of transformants. Pbrab5b-HA fragment was released from pJet1.2 backbone through digestion with HindIII (New England biolabs, Massachusetts, USA) and NotI (New England Biolabs), as per manufacturer's instructions. The Plasmodium expression vector (PEV) (Setua et al., 2020) was also digested with HindIII and NotI. The digests were resolved on 1% agarose gel and the band corresponding to Pbrab5b-HA (1.3 kb) and the linearized PEV (4.9 kb) were excised and purified using the QIAquick Gel Extraction Kit (Qiagen). The sticky-ended DNA fragments were ligated using T4-ligase (New England Biolabs) in a molar ration of 10:1 (insert to vector) as per manufacturer's instructions. The ligation mixture was used to perform bacterial transformation. Transformants were screened by colony PCR (NZYTaq II master mix, Nzytech) and sub-cultured in LB-Ampicillin (100 μg/mL) broth at 37°C, 220 rpm for 16–24 h. Plasmid DNA from transformants were extracted and purified using the NZYprep kit (Nzytech). The Pbrab5b-HA-PEV construct and Pbrab5b_3ÚTR-pJet1.2 construct were digested with HindIII and KpnI followed by resolution on 1% agarose and purification of PbRab5b_3ÚTR band (1.02 kb) and the linearized PbRab5b-HA-PEV vector (6.2 kb). These digested fragments were ligated and transformed as described above. The final cloning vector (7.2 kb) was linearized with HindIII prior to the transfection of P. berghei merozoites. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP (gfp-mu3) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
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 | gfp (FACS) | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration. | ||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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