RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1030100; Gene model (P.falciparum): PF3D7_1412500; Gene product: actin II (ACT2)
Details mutation: Actin II with a point mutation of His73 (his73Q) preventing methylation
Phenotype Oocyst; Sporozoite;
Last modified: 7 March 2023, 14:33
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36877739
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/ResearcherLopez AJ, Kursula I
Name Group/DepartmentDepartment of Biomedicine
Name InstituteUniversity of Bergen
Name of the mutant parasite
RMgm numberRMgm-5278
Principal nameactIIh73q
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNormal numbers of oocysts. However, oocysts were smaller, showed reduced DNA content and lacked sporozoite formation
SporozoiteNo sporozoite formation
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated form of Actin II in which His73 has a point mutation (his73Q) preventing methylation

Protein (function)
Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and have also nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the zygote. The protein is highly expressed in male gametes/gametocytes.

Unlike H74 in actin I, around 90% of actin II has a H73 methylation when expressed in insect cells. Methylation of this histidine stabilizes the filament. To confirm that H73 is indeed methylated in vivo, mass spectrometry was carried out on extracts from zygotes and this revealed a signal consistent with methylation. 
To abolish the regulation by methylation, we introduced a point mutation in the actin II gene, obtaining the actIIH73Q mutant. This mutant was able to normally form male gametes, as exflagellation took place and ookinete conversion was comparable to the WT. Thus, a histidine at position 73 and its possible methylation are not important for the function of actin II in these early mosquito stages. Next, mice infected with WT and the mutant were offered to A. gambiae female mosquitoes. After 12-13 days, midguts were dissected and labeled for the oocyst capsule protein Cap380, and DNA was stained. All oocysts were counted, even those that were small, and this revealed only a minor decrease in the number of oocysts in the actIIH73Q strain comparing to the WT. However, the mutant oocysts were significantly smaller than the WT. In addition, 80% of WT oocysts contained DNA, while only 42% of the mutant oocysts had visible DNA staining. In the mutant oocysts, no sporozoites were formed 

Additional information


Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1030100
Gene Model P. falciparum ortholog PF3D7_1412500
Gene productactin II
Gene product: Alternative nameACT2
Details of the genetic modification
Short description of the mutationActin II with a point mutation of His73 (his73Q) preventing methylation
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPoint mutation H73Q was generated using the QuikChange II Site-directed mutagenesis kit from Agilent according to the manufacturer’s instructions. The template for the generation by PCR of the desired mutation was the pSD141 plasmid containing the WT actin II locus (see RMgm-985). The plasmid was sequenced to verify the correct mutation.
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