RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4345
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PbANKA_0112500; Gene model (P.falciparum): PF3D7_0614300; Gene product: major facilitator superfamily-related transporter, putative, putative (MFR1)
Name tag: triple-HA
Phenotype Asexual bloodstage;
Last modified: 19 September 2017, 17:58
  *RMgm-4345
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28902970
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/ResearcherSayers CP, McFadden GI, Goodman CD
Name Group/DepartmentSchool of BioSciences
Name InstituteUniversity of Melbourne
CityParkville, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4345
Principal nameHA-PbANKA_0112500
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNon-apicoplast location of the protein in the cytoplasm of blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of PbANKA_0112500

Protein (function)
The protein was identified in a bioinformatic screen for identifying apicoplast proteins.
27 candidate apicoplast membrane proteins were identified, 20 of which are annotated as putative membrane transporters.

Candidates were selected from P. falciparum putative membrane transporters belonging to known transport families and selected members of the predicted P. falciparum apicoplast proteome with multiple TMDs. Many P. falciparum membrane transporters have seven or more TMDs, whilst some have six or fewer. Without biological data to confirm membrane transport activity,  candidate proteins predicted to have at least six TMDs were selected. Although multiple TMDs is a minimum requirement of most membrane transporters, some of these proteins may be integral membrane proteins with other functions.

A genetic screen was performed in P. berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast-localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4) and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast-localised.Three apicoplast-localised candidates lack a canonical apicoplast targeting signal but do contain conserved N-terminal tyrosines with likely roles in targeting.

Sixteen candidate genes were HA-tagged for localisation experiments in P. berghei.
Immunofluorescence assays (IFAs) were performed for all tagged candidate genes. Five candidates co-localised with the apicoplast marker, ACP. PbANKA_0809500, PbANKA_0401200, PbANKA_0505500 were all predicted to have apicoplast targeting leader sequences whilst PbANKA_1103600, PbANKA_1304700  were not predicted to contain leader sequences. Another seven candidates localised to non-apicoplast structures within the parasite. Four candidates had no observable immunofluorescence signal at the blood stage so could not be localised in this life cycle stage: PbANKA_0942100, PbANKA_0417100, PbANKA_1446100 and PbANKA_0602400. Life cycle analysis of two candidates (PbANKA_1103600 and PbANKA_0942100) revealed that PbANKA_1103600 TAG was expressed and localised to the apicoplast in midgut oocysts, salivary gland sporozoites and in vitro liver cell stage parasites. No signal was observed for PbANKA_0942100 TAG parasites at any life cycle stage so its localisation remains unknown

Phenotype
Evidence (IFA) is presented for a non-apicoplast location of the protein in the cytoplasm of blood stages.

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_0112500
Gene Model P. falciparum ortholog PF3D7_0614300
Gene productmajor facilitator superfamily-related transporter, putative, putative
Gene product: Alternative nameMFR1
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6