Summary

RMgm-167
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1212600; Gene model (P.falciparum): PF3D7_1014200; Gene product: male gamete fusion factor HAP2, putative (HAP2; GCS1, Generative Cell Specific 1)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 28 January 2011, 14:06
  *RMgm-167
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) : 18367645
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-155
Other information parent lineA gfp-tagged version of hap2 is introduced into a mutant (RMgm-155) that lacks the endogeneous hap2 gene.
The mutant parasite was generated by
Name PI/ResearcherY. Liu; O. Billker
Name Group/DepartmentDivision of Cell and Molecular Biology
Name InstituteImperial College London
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-167
Principal nameHAP2-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteGametocytes express the fusion protein HAP2-GFP.
Complementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in restoration of fertilisation, resulting in fertilisation rates that are comparable to wild type.
Fertilization and ookineteComplementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in normal ookinete production, comparable to wild type.
OocystNot different from wild type
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP-tagged form of HAP2 (GCS1,Generative Cell Specific 1) . The hap2-gfp gene is under the regulation of the endogenous hap2 gene promoter

Protein (function)
In Arabidopsis the male-specific sterility protein HAP2 has been identified by a genetic screen (HAP mutants: containing haploid-disrupting (hapless) mutations; Johnson M.A. et al., 2004, Genetics 168, 971-82) . A HAP2 family member called GCS1 (for generative cell-specific) was subsequently identified in a screen for lily genes whose transcripts were up-regulated in sperm (generative cells) . HAP2 is conserved and members were found in rice, Chlamydomonas, a red alga, a slime mold, Plasmodium falciparum, and Leishmania major.
Phenotype analyses of mutants lacking expression of HAP2/GCS1  (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse.

Phenotype
Gametocytes express the fusion protein HAP2-GFP.
The Complementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in restoration of fertilisation, resulting in normal fertilisation rates and production of ookinetes, comparable to wild type.
Phenotype analyses of mutants lacking expression of HAP2/GCS1  (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse.

Additional information
GenBank accession no. XM_671808.

Other mutants
RMgm-155: An independent mutant lacking expression of HAP2/GCS1.
RMgm-156: A mutant lacking expression of HAP2/GCS1.
RMgm-157: A mutant expressing a GFP-tagged form of HAP2/GCS1
RMgm-158: A mutant expressing GFP under the control of the hap2/gcs1 promoter
RMgm-605: A mutant expressing a mutated form of HAP2/GCS1 lacking the HAP2-GCS1 (HG) domain
RMgm-606: A mutant expressing a mutated form of HAP2/GCS1 lacking the entire C-terminal region downstream from the TM domain
RMgm-607: A mutant expressing a mutated form of HAP2/GCS1 lacking the TM domain and the entire C-terminal region downstream from the TM domain


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1212600
Gene Model P. falciparum ortholog PF3D7_1014200
Gene productmale gamete fusion factor HAP2, putative
Gene product: Alternative nameHAP2; GCS1, Generative Cell Specific 1
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: taggingA 4.2-kb fragment comprising 1.5 kb of upstream sequence and the complete hap2 genomic sequence, except the stop codon, was amplified by PCR from genomic DNA using primers 485 (5′-ATATGGTAC CACGCTACTTATATATAGTGATAACC-3′) and 482 (5′-ATA TGGGCCCTCGCAATGGGGGTATTTTACTTTTAC-3′), inserted into KpnI and ApaI restriction sites of vector p277, upstream of and in frame with an egfp gene, which was followed by 0.5 kb of 3′ untranslated region (UTR) derived from the Pbdhfr/ts gene. The vector was linearized in a unique EcoRV site in the putative hap2 promoter and introduced into a hap2 knockout mutant (RMgm-155).
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single 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
Promoter of the selectable markereef1a
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationA 4.2-kb fragment comprising 1.5 kb of upstream sequence and the complete hap2 genomic sequence, except the stop codon, was amplified by PCR from genomic DNA using primers 485 (5′-ATATGGTAC CACGCTACTTATATATAGTGATAACC-3′) and 482 (5′-ATA TGGGCCCTCGCAATGGGGGTATTTTACTTTTAC-3′), inserted into KpnI and ApaI restriction sites of vector p277, upstream of and in frame with an egfp gene, which was followed by 0.5 kb of 3′ untranslated region (UTR) derived from the Pbdhfr/ts gene. The vector was linearized in a unique EcoRV site in the putative hap2 promoter and introduced into a hap2 knockout mutant (RMgm-155).
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