RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4151
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1442200; Gene model (P.falciparum): PF3D7_1227500; Gene product: protein SOC2, putative (SOC2)
Name tag: triple-HA
Transgene
Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1319500; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (LAP4; LCCL/lectin adhesive-like protein 4; CCp2)
3'UTR: Gene model: PBANKA_1359600; Gene product: transmission blocking target antigen precursor 6-cysteine protein (P48/45)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_0416100; Gene model (P.falciparum): PF3D7_0905300; Gene product: dynein heavy chain, putative
3'UTR: Gene model: PBANKA_1010600; Gene product: calmodulin, putative (cam)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete;
Last modified: 22 May 2017, 15:38
  *RMgm-4151
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28481199
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 820cl1m1cl1 (RMgm-164)
Other information parent lineP. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517).
The mutant parasite was generated by
Name PI/ResearcherFang H, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4151
Principal nameSOC2-3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteC-terminal 3xHA tagging of SOC2 only allowed detection of a c.a. 140 kDa protein. Immunoprecipitation of SOC2-3xHA recovered peptides mapping on the last 1,137 amino acids suggesting SOC2 is processed into at least two different isoforms. The 3xHA tagged C-terminal end of SOC2-3xHA was found to colocalise with mitotic spindles in activated male gametocytes suggesting a role in their assembly.
Normal exflagellation.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 3xHA-tagged version of SOC2 and expresses RFP and GFP in female and male gametocytes, respectively.

Protein (function)
The protein is identified in a screen for proteins that are phosphorylated by CDPK4.CDPK4 has an essential role in male (micro)gamete formation (exflagellation).
Among these proteins, it was decided to name those without predicted function SOC, for substrate of CDPK4. Ten proteins were indeed conserved Plasmodium proteins of unknown function and one  protein, SOC2, was annotated as cyclin-related protein 2 but it was suggested be unrelated to cyclins. Also GAP40 was identified.
To investigate the roles of SOC1 to 4 and GAP40 during gametogenesis, it was attempted to individually  knock-out their encoding proteins. As controls, soc5 and soc6 were included that were not differentially phosphorylated in a CDPK4-KO mutant. For gap40 no transgenic parasites could be obtained. Viable KO parasites could be detected in mixed asexual stages for six genes and non-clonal populations were first assessed for microgametocyte DNA replication and exflagellation. No defects were observed in the SOC4-KO line as well as in the SOC5-KO and SOC6-KO control lines suggesting that these proteins do not represent crucial CDPK4 effectors in the regulation of male gametogenesis. Defects in DNA replication or exflagellation were observed for SOC1-KO, SOC2-KO, and SOC3-KO lines that were cloned for in depth characterisation. 

Phenotype
C-terminal 3xHA tagging of SOC2 only allowed detection of a c.a. 140 kDa protein. Immunoprecipitation of SOC2-3xHA recovered peptides mapping on the last 1,137 amino acids suggesting SOC2 is processed into at least two different isoforms. The 3xHA tagged C-terminal end of SOC2-3xHA was found to colocalise with mitotic spindles in activated male gametocytes suggesting a role in their assembly

Phenotype analyses of a mutant lacking expression of SOC2 (RMgm-4145) showed the following: Reduced growth/multiplication of asexual blood stages. Strongly reduced exflagellation (male gamete formation). Reduced DNA replication during male gamete formation (mainly blocked at 2N; proportions of haploid microgametocytes in 10 min activated microgametocytes were similar in the KO and its WT counterpart indicating that SOC2 is not required for initiation of DNA replication and DNA replication per se. However, 47% of the parasites remained diploid and 20% and 16% reached the 4N and 8N levels, respectively. Strongly reduced mitotoic spindle formation in microgametocytes at 10 min. after activation. Normal axoneme in microgametocytes at 10 min. after activation..

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1442200
Gene Model P. falciparum ortholog PF3D7_1227500
Gene productprotein SOC2, putative
Gene product: Alternative nameSOC2
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 usedNo
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameRFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1319500
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP4; LCCL/lectin adhesive-like protein 4; CCp2
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_1359600
Gene producttransmission blocking target antigen precursor 6-cysteine protein
Gene product: Alternative nameP48/45
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mut3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0416100
Gene Model P. falciparum ortholog PF3D7_0905300
Gene productdynein heavy chain, putative
Gene product: Alternative name
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_1010600
Gene productcalmodulin, putative
Gene product: Alternative namecam
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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 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