RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5029
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1437500; Gene model (P.falciparum): PF3D7_1222600; Gene product: AP2 domain transcription factor AP2-G (AP2-G; ApiAP2)
Name tag: EGFP
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 7 July 2021, 11:44
  *RMgm-5029
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) : 34119684
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/ResearcherYuda M, Nishi T
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteMie University School of Medicine
CityMie, Tsu
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5029
Principal nameAP2-G-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageFluorescent signals, indicating AP2-G expression, were detected only in a very small number of mononuclear parasites by fluorescence microscopy (one parasite per dozens of fields by microscopic scrutiny). We assumed that they were early gametocytes and that their number appeared very small, as AP2-G is expressed only in a limited period of gametocyte development.
In synchronized infections: In mature schizonts prior to inoculation, AP2-G expression was not observed. At 12 h post-inoculation (hpi), weak signals were first observed in a small number of parasites. Signals of AP2-G and the pro-portion of AP2-G-expressing parasites increased gradually thereafter (the proportion at 14 hpi was approximately 2%) and then reached the peak at 16 to 18 hpi, when morphological divergence between early gametocytes and asexual parasites began. The proportion of AP2-G- expressing parasites was approximately 4% at this peak. Then, fluorescent signals decreased gradually, and at 20 to 22 hpi, when sex-specific morphologies were manifested on them, signals decreased to the level where AP2-G was only slightly observed in the nucleus
Gametocyte/GameteFluorescent signals, indicating AP2-G expression, were detected only in a very small number of mononuclear parasites by fluorescence microscopy (one parasite per dozens of fields by microscopic scrutiny). We assumed that they were early gametocytes and that their number appeared very small, as AP2-G is expressed only in a limited period of gametocyte development.
In synchronized infections: In mature schizonts prior to inoculation, AP2-G expression was not observed. At 12 h post-inoculation (hpi), weak signals were first observed in a small number of parasites. Signals of AP2-G and the pro-portion of AP2-G-expressing parasites increased gradually thereafter (the proportion at 14 hpi was approximately 2%) and then reached the peak at 16 to 18 hpi, when morphological divergence between early gametocytes and asexual parasites began. The proportion of AP2-G- expressing parasites was approximately 4% at this peak. Then, fluorescent signals decreased gradually, and at 20 to 22 hpi, when sex-specific morphologies were manifested on them, signals decreased to the level where AP2-G was only slightly observed in the nucleus
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal (enhanced) GFP-tagged version of AP2-G 

Protein (function)
PbAP2-G belongs to the 27 Plasmodium ApiAP2 family of transcription factors

Phenotype
Fluorescent signals, indicating AP2-G expression, were detected only in a very small number of mononuclear parasites by fluorescence microscopy (one parasite per dozens of fields by microscopic scrutiny). We assumed that they were early gametocytes and that their number appeared very small, as AP2-G is expressed only in a limited period of gametocyte development.

In synchronized infections: In mature schizonts prior to inoculation, AP2-G expression was not observed. At  12  h  post-inoculation (hpi), weak signals were first observed in a small number of parasites. Signals of AP2-G and the pro-portion of  AP2-G-expressing parasites increased gradually thereafter (the proportion at 14 hpi was approximately 2%) and then reached the peak at 16 to 18 hpi, when morphological divergence between early gametocytes and asexual parasites began. The proportion of  AP2-G- expressing parasites was approximately 4% at this peak. Then, fluorescent signals decreased gradually, and at 20 to 22 hpi, when sex-specific morphologies were manifested on them, signals decreased to the level where AP2-G was only slightly observed in the nucleus

Additional information
It was supposed that expression level of eGFP was too low to detect with eGFP and therfore another transgenic parasite was prepared by exchanging GFP with mNeonGreen, a  monomeric yellow and green fluorescent protein that has been reported to emit signals approximately three times brighter than GFP.  However, expression was still observed only in the nuclei of small mononuclear parasites, and in a very small number of cells.

From the Abstract:
'Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in  early gametocytes. Expression of  AP2-G decreased with manifestation of  sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of  these genes resulted in  the arrest of  ookinete development. These results suggest another role of  AP2-G: activating a  transcriptional cascade to  promote conversion into early gametocytes.'

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1437500
Gene Model P. falciparum ortholog PF3D7_1222600
Gene productAP2 domain transcription factor AP2-G
Gene product: Alternative nameAP2-G; ApiAP2
Details of the genetic modification
Name of the tagEGFP
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
Promoter of the selectable markerunknown
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