RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1105300; Gene model (P.falciparum): PF3D7_0505700; Gene product: conserved Plasmodium membrane protein, unknown function (akratin)
Details mutation: P. berghei akratin gene replaced with P. falciparum akratin gene with a GFP tag
PhenotypeNo phenotype has been described
Last modified: 28 October 2020, 18:23
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4885
Other information parent lineIn this mutant the akarin gene has been deleted. This mutant does not contain a drug selectable marker (SM). The SM has been removed by negative selection
The mutant parasite was generated by
Name PI/ResearcherKehrer J, Frischknecht F
Name Group/DepartmentCenter for Infectious Diseases, Integrative Parasitology
Name InstituteHeidelberg University Medical School
Name of the mutant parasite
RMgm numberRMgm-4887
Principal nameP. falciparum akratin::gfp
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
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot tested
Additional remarks phenotype

In the mutant the endogenous P. berghei akratin gene has been replaced with the P. falciparum akratin gene that is C-terminally GFP-tagged. The GFP-tagged P. falciparum akratin gene has been introduced into the akratin locus of a mutant (see RMgm-4885) in which the akratin gene has been deleted.
Published in bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318857.

Protein (function)
PbANKA_1105300 was found to be conserved among Plasmodium spp. It shares 82% identity with its orthologue in P. yoelii, but only 35% and 32% with its orthologues in P. falciparum and P. vivax, respectively. In contrast to P. berghei and P. falciparum, the orthologues in P. yoelii and P. vivax are predicted to contain only two TMDs. Furthermore, the P. falciparum protein contains about two times more amino acids than the P. berghei protein. No orthologue was found outside Plasmodium spp

A mutant lacking expression of akratin (see mutant RMgm-4885) fails to produce male gametes (and ookinetes and oocysts)

The normal production of ookinetes and oocysts of mutants expressing P. falciparum akratin shows that the P. falciparum orthologue is  functional in P. berghei even though it has a long N-terminal extension and an overall identity of only 35%

Additional information
In this study also a mutant has been generated that expresses a C-terminal GFP-tagged version of P. berghei akratin. The following evidence is presented on akratin expression and localisation:
'We next investigated the localization of the fluorescent signal in the blood stage and the extracellular forms of the life cycle. This revealed a mostly diffuse signal with some punctae in blood stages of both parasite lines expressing the P. falciparum or P. berghei akratin-GFP. In non-activated gametocytes the signal was distributed within the gametocyte cytoplasm but it was much weaker in the P. falciparum akratin-GFP line, where it appeared punctate. No difference between male and female gametocytes could be observed. However, upon activation two distinct populations, most likely males and females could be observed. While in females the protein was distributed in the cytoplasm of the cell, male (determined by a cloudy Hoechst staining) showed one bright spot in addition to a few weak dots at the periphery. Ookinetes showed a vesicular/endomembranous staining with a prominent peripheral staining for the P. berghei akratin-GFP but an exclusively vesicular pattern for the P. falciparum akratin- GFP. Sporozoites isolated from the oocysts or salivary glands showed a similar punctate localization for both lines reminiscent of secretory vesicles but distinct from the strong apical signal seen in GFP-TRAP sporozoites 

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1105300
Gene Model P. falciparum ortholog PF3D7_0505700
Gene productconserved Plasmodium membrane protein, unknown function
Gene product: Alternative nameakratin
Details of the genetic modification
Short description of the mutationP. berghei akratin gene replaced with P. falciparum akratin gene with a GFP tag
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 parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 5`UTR together with the entire ORF of PbANKA_1105300 was amplified from wt gDNA using primers JK66 and JK152 and inserted into the pL28 plasmid using KpnI and NdeI leading to the replacement of the selection marker. A TgDHFR selection cassette was amplified using primers JK153 and JK154 and inserted between the GFP and 3`UTR using NotI and EcorV resulting in plasmid pL59.
The 5`UTR together with the entire ORF was amplified from wt gDNA using primers JK66 and a reverse Primer introducing the mutations and inserted into the pL59 plasmid using KpnI and NdeI resulting in pL84.
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