Summary

RMgm-1341
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1034300; Gene model (P.falciparum): PF3D7_1408200; Gene product: AP2 domain transcription factor AP2-G2, putative (AP2-G2; ApiAP2)
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; Fertilization and ookinete; Oocyst;
Last modified: 25 October 2015, 13:02
  *RMgm-1341
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26417110
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/ResearcherYuda M; Kato T
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteMie University School of Medicine
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-1341
Principal nameAP2-G2(−)820
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNo mature male or female gametocytes are formed. No exflagellation. Evidence is presented that sexually committed parasites (trophozoites) are formed within red blood cells but that these parasites abort development before morphological features of sexual differentiation are visible at the light-microscopic level.
Fertilization and ookineteNo mature male or female gametocytes are formed. No exflagellation. No oocysts are formed.
OocystNo mature male or female gametocytes are formed. No exflagellation. No oocysts are formed.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of AP2-G2. In addition, it expresses GFP in male gametocytes and RFP in female gametocytes.

Protein (function)
Apetala 2 (AP2)-family proteins are transcription factors that have DNA-binding domains of 60 amino acids called AP2 domains. Recently, AP2 genes have been found in the genomes of Plasmodium parasites. In P. falciparum 27 AP2-family genes have been identified. Among these genes, 26 are conserved in the other Plasmodium species whose entire genomes have been sequenced. Each member of this family has 1 to 4 AP2 domains, and the amino acid sequences of these domains are highly conserved among Plasmodium orthologs.

Phenotype
No mature male or female gametocytes are formed. No exflagellation. Evidence is presented that sexually committed parasites (trophozoites) are formed within red blood cells but that these parasites abort development before morphological features of sexual differentiation are visible at the light-microscopic level.

Evidence is presented that the lack of AP2-G2 expression did not abrogate sexual commitment or sex determination but did cause marked developmental defects in the gametocytes, together with a marked reduction of sex-specific gene expression.

Evidence is presented that AP2-G2 is a transcriptional repressor in sexually committed trophozoites. Evidence is presented that most of these target genes of AP2-G2 binding are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program of asexual replication to promote conversion to the sexual stage. These 'asexual' genes were upregulated in the parasites lacking expression of AP2-G2

Additional information
The AP2-G2(−)820 parasites showed the same phenotype observed in the AP2-G2(−) populations prepared from wild-type parasites (RMgm-1340); i.e., they produced no mature gametocytes. Based on flow cytometric analysis, the signal strengths of both fluorescent proteins in AP2-G2(−)820 parasites were markedly lower than in the original parasites. However, the ratio of RFP-positive to GFP-positive cells was similar between AP2-G2(−) parasites and the original parasites (0.95 and 1.16, respectively), and the two populations presented no overlap with each other, indicating that AP2-G2(–)820 parasites commit to one sex at a sex ratio similar to that in the original 820cl1m1cl1 parasites.

In analyses using mutant P. berghei parasites expressing GFP-tagged AP2-G2 (AP2-G2::GFP parasites; RMgm-1342), it was found that AP2-G2 was specifically expressed in female and male gametocytes. The GFP signal was solely observed in the nucleus, suggesting that AP2-G2 acts as a transcription factor in gametocytes. In a temporal profiling analysis of AP2-G2 expression using synchronized blood cultures, expression was first observed from 16 h after erythrocyte invasion (hpi) until development into mature male or female gametocytes. The timing of expression correlated well with the onset of morphological divergence from the asexual stage (16–18 hpi), strongly suggesting that this transcription factor is involved in gametocyte-specific gene expression.

Other mutants
See link for other AP2-G2 mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1034300
Gene Model P. falciparum ortholog PF3D7_1408200
Gene productAP2 domain transcription factor AP2-G2, putative
Gene product: Alternative nameAP2-G2; ApiAP2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
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 15´-ACCACGATAAAGGAGGCATGTAAC-3´
Additional information primer 1AP2-G2-1
Sequence Primer 25´-CTCATCTACAAGCATCgtcgacTCATCGTCTCCTTTCCTC-3´
Additional information primer 2AP2-G2-2
Sequence Primer 35´-CCTTCAATTTCGgatccactagGTAATGTTGAAAGCGACAG-3´
Additional information primer 3AP2-G2-3
Sequence Primer 45´-GTTGTATTATCAACTTGAGCAGTTTC-3´
Additional information primer 4AP2-G2-4
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