RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-790
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0915600; Gene model (P.falciparum): PF3D7_1132800; Gene product: aquaglyceroporin (AQP)
Name tag: mCherry + cmyc (3x)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_0915600; Gene product: aquaglyceroporin (AQP)
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 20 August 2013, 10:49
  *RMgm-790
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherS. Kenthirapalan; T.W. Kooij
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-790
Principal nameaqp::tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagemCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes)
Gametocyte/GametemCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes)
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry/cmyc-tagged version of AQP and expresses GFP under the control of the constitutive and strong hsp70 promoter.
The construct to tag the aqp gene contains a GFP expression cassette and a tag-sequence. The tag consists of mCherry and a triple cmyc epitope. 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)
The mutant parasite is cloned by FACS sorting (and not by limiting dilution) based on GFP expression.

Protein (function)
Aquaporins constitute a family of cellular water and solute channels. They are functionally divided into two groups, the aquaporins, which are water-specific channels, and aquaglyceroporins, which are highly permeated by glycerol and other solutes and variably permeated by water. Aquaglyceroporins are the only glycerol channels identified in mammals.

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.

Phenotype
Phenotype analyses of mutants (RMgm-265, RMgm-789) lacking expression of AQP during blood, mosquito and liver stage parasites indicate that AQP is not essential during the complete life cycle. 

mCherry-tagged AQP was found to be localized in the cytoplasm of blood stages (asexual stages and gametocytes)

Additional information
See also mutant RMgm-265. Analysis of localisation of AQP using antibodies indicated a location at the plasmamembrane of blood stages

Other mutants
RMgm-265 - A mutant lacking expression of AQP
RMgm-789 - An independent mutant lacking expression of AQP


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0915600
Gene Model P. falciparum ortholog PF3D7_1132800
Gene productaquaglyceroporin
Gene product: Alternative nameAQP
Details of the genetic modification
Name of the tagmCherry + cmyc (3x)
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ScaI, SalI
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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 procedureThe mutant parasite is cloned by FACS sorting (and not by limiting dilution) based on GFP expression.
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1AATCCTAGGCATTATTATAAAATATATTTGTGAGACTG
Additional information primer 1AQP-F5-AvrII; 3' untranslated region
Sequence Primer 2ATAGGTACCTTGCAAATCTGTCTTGTCGTC
Additional information primer 2AQP-R4-KpnI; 3' untranslated region
Sequence Primer 3TAACCGCGGTATTTTGCAGGACAACTCCTTG
Additional information primer 3AQP-F4-SacII; 527 bp fragment of the C-
terminal coding region of AQP
Sequence Primer 4TAAGACATATGTCCTATTTCTAAGGCGCCTTTATCATG
Additional information primer 4AQP-R3-PshAI; 527 bp fragment of the C-
terminal coding region of AQP
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ScaI, SalI
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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 procedureThe mutant parasite is cloned by FACS sorting (and not by limiting dilution) based on GFP expression.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0915600
Gene productaquaglyceroporin
Gene product: Alternative nameAQP
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1AATCCTAGGCATTATTATAAAATATATTTGTGAGACTG
Additional information primer 1AQP-F5-AvrII; 3' untranslated region
Sequence Primer 2ATAGGTACCTTGCAAATCTGTCTTGTCGTC
Additional information primer 2AQP-R4-KpnI; 3' untranslated region
Sequence Primer 3TAACCGCGGTATTTTGCAGGACAACTCCTTG
Additional information primer 3AQP-F4-SacII; 527 bp fragment of the C-
terminal coding region of AQP
Sequence Primer 4TAAGACATATGTCCTATTTCTAAGGCGCCTTTATCATG
Additional information primer 4AQP-R3-PshAI; 527 bp fragment of the C-
terminal coding region of AQP