SummaryRMgm-790
|
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) : 23137753 |
MR4 number | |
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 | Not applicable |
Other information parent line | |
top of page | |
The mutant parasite was generated by | |
Name PI/Researcher | S. Kenthirapalan; T.W. Kooij |
Name Group/Department | Parasitology Unit |
Name Institute | Max Planck Institute for Infection Biology |
City | Berlin |
Country | Germany |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-790 |
Principal name | aqp::tag |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
top of page | |
Phenotype | |
Asexual blood stage | mCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes) |
Gametocyte/Gamete | mCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes) |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phylogenetic analysis, in comparison with representative AQPs of human and Arabidopsis thaliana, revealed a strong association of Plasmodium AQP (PBANKA_091560; PF3D7_1132800) with human type II aquaporins and, more distantly, with the A. thaliana nodulin-like intrinsic protein (NIP) family. Toxoplasma gondii AQP1 (TGME49_015450) clusters with the second, unusually long, predicted Plasmodium AQP (PBANKA_142710; PF3D7_0810400) and associates more closely to a clade containing the A. thaliana small basic intrinsic protein (SIP) family members and humanAQP11 and 12. Aquaporins are characterized by two ‘‘NPA’’ boxes that are relatively well conserved across most subfamilies. Sequence alignments of these motifs of P. berghei and P. falciparum AQP1 with selected aquaporin sequences, i.e. human AQP3, 7, 9, and 10 and the A. thaliana NIP subfamily members, reveals significant conservation but also some striking differences. PbAQP1 and PfAQP1 are both marked by unusual variations in the invariant ‘‘NPA’’ sequences. Furthermore, a highly conserved proline in the second ‘‘NPA’’ box is absent in both malaria parasites. mCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes) |
top of page | |||||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0915600 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1132800 | ||||||||||||||||||||||||||
Gene product | aquaglyceroporin | ||||||||||||||||||||||||||
Gene product: Alternative name | AQP | ||||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | mCherry + cmyc (3x) | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
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 | ScaI, SalI | ||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The construct to tag the aqp gene contains a GFP expression cassette. In this cassette the gfp gene is under the control of the hsp70 promoter (see for more information on the basic plasmid pBAT-SIL6 the mutant RMgm-757). Parasites with their AQP C-terminally tagged were generated using the standard gene replacement strategy. First, a 526 bp fragment of the 3' untranslated region (UTR) was amplified by PCR from genomic DNA using the primer combination AQP-F5-AvrII and AQP-R4-KpnI and cloned into the pBAT-SIL6 vector (Kooij et al., 2012) using AvrII and KpnI restriction digestion to generate the intermediate construct pAQP-IM. For the tagging construct, pAQP-tag, a 527 bp fragment of the C-terminal coding region of AQP was amplified from gDNA using the gene-specific primers, AQP-F4-SacII and AQP-R3-PshAI, and fused in frame to the mCherry-3xMyc tag of pAQP-IM using SacII and HpaI. The resulting plasmid was linearized with ScaI and SalI and transfected into WT P. berghei ANKA strain parasites. | ||||||||||||||||||||||||||
Additional remarks selection procedure | The mutant parasite is cloned by FACS sorting (and not by limiting dilution) based on GFP expression. | ||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
top of page |
top of page | |||||||||||||||||||
Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP | ||||||||||||||||||
top of page | |||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
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 | ScaI, SalI | ||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The construct to delete the aqp gene contains a GFP expression cassette. In this cassette the gfp gene is under the control of the hsp70 promoter (see for more information on the basic plasmid pBAT-SIL6 the mutant RMgm-757). Parasites with their AQP C-terminally tagged were generated using the standard gene replacement strategy. First, a 526 bp fragment of the 3' untranslated region (UTR) was amplified by PCR from genomic DNA using the primer combination AQP-F5-AvrII and AQP-R4-KpnI and cloned into the pBAT-SIL6 vector (Kooij et al., 2012) using AvrII and KpnI restriction digestion to generate the intermediate construct pAQP-IM. For the tagging construct, pAQP-tag, a 527 bp fragment of the C-terminal coding region of AQP was amplified from gDNA using the gene-specific primers, AQP-F4-SacII and AQP-R3-PshAI, and fused in frame to the mCherry-3xMyc tag of pAQP-IM using SacII and HpaI. The resulting plasmid was linearized with ScaI and SalI and transfected into WT P. berghei ANKA strain parasites. | ||||||||||||||||||
Additional remarks selection procedure | The mutant parasite is cloned by FACS sorting (and not by limiting dilution) based on GFP expression. | ||||||||||||||||||
top of page | |||||||||||||||||||
Other details transgene | |||||||||||||||||||
top of page | |||||||||||||||||||
Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
| |||||||||||||||||||
top of page | |||||||||||||||||||
3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1340000 | ||||||||||||||||||
Gene product | dihydrofolate synthase/folylpolyglutamate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | PbDHFS-FPGS | ||||||||||||||||||
| |||||||||||||||||||
Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0915600 | ||||||||||||||||||
Gene product | aquaglyceroporin | ||||||||||||||||||
Gene product: Alternative name | AQP | ||||||||||||||||||
| |||||||||||||||||||
top of page |