SummaryRMgm-4656
|
Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
The following genetic modifications were attempted | Gene mutation, Introduction of a transgene |
Number of attempts to introduce the genetic modification | Unknown |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 31364224 |
top of page | |
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 | P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
top of page | |
Attempts to generate the mutant parasite were performed by | |
Name PI/Researcher | Wolanin K, Mueller AK, Prudencio M |
Name Group/Department | Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina |
Name Institute | Universidade de Lisboa |
City | Lisbon |
Country | Portugal |
top of page | |||||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0926700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1121600 | ||||||||||||||||||||||||||
Gene product | exported protein 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | HEP17, EXP1 | ||||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | N-terminal(NT)-domain truncation of PbEXP1 | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system |
The unsuccessful attempts to truncate the N-terminal portion of EXP1 indicate an essential function of this region during asexual blood stage growth. Exp1 (HEP17) is a protein that localizes to the parasitophorous vacuole membrane (PVM) of blood stages and liver stages Pb/PfEXP1’s topology includes an N-terminal (NT) domain (aa 1-75/1-79) that harbors a classical signal peptide (aa 1-23); a putative catalytic domain that includes the Pf Arg70 residue and is presumed to be responsible for GST activity in blood stage parasites; an internal hydrophobic region typical of transmembrane anchor sequences (aa 75-97/79-101); and a C-terminal (CT) domain (aa 97-166/101-162) that includes a C1 (aa 103-136/107-134) and a C2 (aa 137-166/135-162) region, as defined in (Sa et al., 2017). In both blood and liver parasite stages, EXP1 is proposed to be integrated into the PVM with the N-terminus facing the lumen of the PV, and the C-terminus exposed to the host cell cytosol. In order to dissect the functional role of different domains of PbEXP1, we attempted to generate modified versions of this protein bearing a truncated version of its N- and C-terminal domains. Despite multiple attempts, we were unable to generate a modified version of PbEXP1 lacking the NT domain (PbEXP1ΔNT; RMgm-4656), suggesting that it is essential for blood stage development of Pb parasites. However, transgenic Pb parasite lines expressing a CT domain-truncated version of PbEXP1 were successfully generated in both the PbANKA (PbWT) and PbGFPcon (PbGFP) parental parasite lines, yielding their PbEXP1ΔCT and PbGFPEXP1ΔCT mutated counterparts, respectively, and showing that Pb blood stages can develop in the absence of the PbEXP1 CT domain. Successful deletion of the CT domain in the PbEXP1ΔCT parasites was confirmed by Western blot using two different anti-EXP1 antibodies that bind either to epitopes present on the full length PbEXP1 protein or to epitopes present only on the protein’s C-terminus. | ||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate the PbGFPEXP1ΔCT parasite lines, a 3’ UTR fragment was amplified by using 3’UTR PbEXP1for and 3’UTR PbEXP1rev primers, and a 5’ fragment including the ORF without the last 207 bp of PbEXP1 was amplified with 5’UTR PbEXP1 ORF dC2for (Sa et al., 2017) and 5’UTR PbEXP1 ORF dCTrev primers from PbWT genomic DNA (gDNA) and cloned into the b3D+ vector (Silvie, Goetz, & Matuschewski, 2008). A similar strategy was used to generate the PbEXP1ΔNT parasite line. Briefly, a 5′ fragment without the last 114 bp coding N-terminal domain of EXP1 protein after signal peptide was amplified using primers 5’UTR SP PbEXP1for and 5’UTR SP PbEXP1rev together with full transmembrane domain (TM) containing C-terminal domain (CT) using primers TM CT PbEXP1for and TM CT PbEXP1rev from PbWT gDNA and cloned into the b3D+ vector containing the 3′ UTR fragment as described above. Before transfection, the targeting vectors were linearized by using restriction enzymes KpnI and NotI. The unsuccessful attempts to truncate the N-terminal portion of EXP1 indicate an essential function of this region during asexual blood stage growth. Exp1 (HEP17) is a protein that localizes to the parasitophorous vacuole membrane (PVM) of blood stages and liver stages Pb/PfEXP1’s topology includes an N-terminal (NT) domain (aa 1-75/1-79) that harbors a classical signal peptide (aa 1-23); a putative catalytic domain that includes the Pf Arg70 residue and is presumed to be responsible for GST activity in blood stage parasites; an internal hydrophobic region typical of transmembrane anchor sequences (aa 75-97/79-101); and a C-terminal (CT) domain (aa 97-166/101-162) that includes a C1 (aa 103-136/107-134) and a C2 (aa 137-166/135-162) region, as defined in (Sa et al., 2017). In both blood and liver parasite stages, EXP1 is proposed to be integrated into the PVM with the N-terminus facing the lumen of the PV, and the C-terminus exposed to the host cell cytosol. In order to dissect the functional role of different domains of PbEXP1, we attempted to generate modified versions of this protein bearing a truncated version of its N- and C-terminal domains. Despite multiple attempts, we were unable to generate a modified version of PbEXP1 lacking the NT domain (PbEXP1ΔNT; RMgm-4656), suggesting that it is essential for blood stage development of Pb parasites. However, transgenic Pb parasite lines expressing a CT domain-truncated version of PbEXP1 were successfully generated in both the PbANKA (PbWT) and PbGFPcon (PbGFP) parental parasite lines, yielding their PbEXP1ΔCT and PbGFPEXP1ΔCT mutated counterparts, respectively, and showing that Pb blood stages can develop in the absence of the PbEXP1 CT domain. Successful deletion of the CT domain in the PbEXP1ΔCT parasites was confirmed by Western blot using two different anti-EXP1 antibodies that bind either to epitopes present on the full length PbEXP1 protein or to epitopes present only on the protein’s C-terminus. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
top of page |
top of page | |||||||||||||||||||
Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP (gfp-mu3) | ||||||||||||||||||
top of page | |||||||||||||||||||
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 | |||||||||||||||||||
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. | ||||||||||||||||||
top of page | |||||||||||||||||||
Other details transgene | |||||||||||||||||||
top of page | |||||||||||||||||||
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 | ||||||||||||||||||
| |||||||||||||||||||
top of page | |||||||||||||||||||
3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
| |||||||||||||||||||
Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
| |||||||||||||||||||
top of page |