RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-908
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0830400; Gene model (P.falciparum): PF3D7_0929600; Gene product: G2 protein, putative (G2)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 21 June 2013, 22:32
  *RMgm-908
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) : 23773015
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherA. Tremp, J.T. Dessens
Name Group/DepartmentDepartment of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon 11 School of Hygiene & Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-908
Principal nameG2::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteVery weak cytoplasmic GFP based fluorescence in female gametocytes.
Fertilization and ookineteVery weak cytoplasmic GFP based fluorescence in female gametocytes. Fluorescence levels increased concomitant with ookinete development culminating in mature ookinetes with strong GFP signal concentrated both at the periphery of the cell and in an apically located cap-like structure with a pore in the centre. Retorts (i.e. developing, immature ookinetes) displayed cortical fluorescence only in the ‘ookinete’ portion that protrudes from the spherical zygote, indicating that G2 associates with the pellicle structure and not the plasma membrane.
OocystVery weak cytoplasmic GFP based fluorescence in female gametocytes. Fluorescence levels increased concomitant with ookinete development culminating in mature ookinetes with strong GFP signal concentrated both at the periphery of the cell and in an apically located cap-like structure with a pore in the centre.
In young oocysts at four days after infecting mosquitoes, GFP fluorescence had largely disappeared, except for the apical cap structure, and at six days post-infection the majority of oocysts lacked any discernible fluorescence. GFP-based fluorescence was again observed in oocysts from around 8-10 days after infecting mosquitoes, reaching peak levels during sporulation. The G2::GFP fusion protein concentrated at the periphery of the sporozoites, but in contrast to ookinetes was found associated with neither anterior nor posterior end. This is consistent with the observation that Plasmodium sporozoites, contrary to ookinetes, do not possess a cap-like structure.
SporozoiteIn young oocysts at four days after infecting mosquitoes, GFP fluorescence had largely disappeared, except for the apical cap structure, and at six days post-infection the majority of oocysts lacked any discernible fluorescence. GFP-based fluorescence was again observed in oocysts from around 8-10 days after infecting mosquitoes, reaching peak levels during sporulation. The G2::GFP fusion protein concentrated at the periphery of the sporozoites, but in contrast to ookinetes was found associated with neither anterior nor posterior end. This is consistent with the observation that Plasmodium sporozoites, contrary to ookinetes, do not possess a cap-like structure.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of G2

Protein (function)
G2 (glycine at position 2) is predicted to be lipid modified on the glycine residue at position 2 through myristoylation, reflected by the presence of a strong canonical amino-terminal myristoylation motif. This protein is orthologous to the ILP1 of Toxoplasma (TGME49_313380).

Phenotype analyses of a mutant lacking expression of G2 (RMgm-907) indicate a role of G2 in morphology and motility of ookinetes and sporozoites, resulting in a loss of infectivity of both ookinetes (reduced oocyst formation) and sporozoites (no invasion of salivary glands).

Phenotype
Phenotype analyses of a mutant lacking expression of G2 (RMgm-907) indicate a role of G2 in morphology and motility of ookinetes and sporozoites, resulting in a loss of infectivity of both ookinetes (reduced oocyst formation) and sporozoites (no invasion of salivary glands).

G2::GFP parasites developed normally in mice and mosquitoes and were readily transmitted by infected mosquito bites, demonstrating that the GFP fusion to the G2 protein did not adversely affect parasite development.

Examination of blood-stage parasites revealed very weak cytoplasmic GFP based fluorescence in female gametocytes. Fluorescence levels increased concomitant with ookinete development culminating in mature ookinetes with strong GFP signal concentrated both at the periphery of the cell and in an apically located cap-like structure with a pore in the centre. Retorts (i.e. developing, immature ookinetes) displayed cortical fluorescence only in the ‘ookinete’ portion that protrudes from the spherical zygote, indicating that G2 associates with the pellicle structure and not the plasma membrane.
In young oocysts at four days after infecting mosquitoes, GFP fluorescence had largely disappeared, except for the apical cap structure, and at six days post-infection the majority of oocysts lacked any discernible fluorescence. GFP-based fluorescence was again observed in oocysts from around 8-10 days after infecting mosquitoes, reaching peak levels during sporulation. The G2::GFP fusion protein concentrated at the periphery of the sporozoites, but in contrast to ookinetes was found associated with neither anterior nor posterior end. This is consistent with the observation that Plasmodium sporozoites, contrary to ookinetes, do not possess a cap-like structure.

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0830400
Gene Model P. falciparum ortholog PF3D7_0929600
Gene productG2 protein, putative
Gene product: Alternative nameG2
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe coding sequence of g2 plus approximately 0.6 kb of the 5′UTR were PCR amplified with primers pDNR-G2-F (ACGAAGTTATCAGTCGACGGTACCATTTTTGGCTAGATTTTATGACTTA) and pDNR-G2-R (ATGAGGGCCCCTAAGCTTATACATAATGAATATTCTTTTTTTTGCC) and introduced into SalI/HindIII-digested pDNR-EGFP via In-Fusion cloning to give plasmid pDNR-G2/EGFP. An approximately 0.7 kb sequence corresponding to the 3′UTR of g2 was PCR amplified with primers pLP-G2-F (TAAACCATTGGTCATAATGTGATGTCTTTCATATGATTCTC) and pLP-G2-R (CGGCCGCTCTAGCATGAAAAGCATATGATGTTATGAAGATG) and introduced into NdeI478 digested pLP-hDHFR2 via In-Fusion cloning to give plasmid pLP-DHFR/G2. The g2-specific sequence from pDNR-G2/EGFP was introduced into pLP-DHFR/G2 via Cre-loxP site-specific recombination to give the final transfection construct pLP-G2/EGFP, used to introduce a GFP-tagged version of g2 into its locus.
Plasmid pDNR-G2/EFGP served as a template for PCR using primers deltaG2-F (ATTGAAAAAAGCTTAGGGGCCCTCAT) and deltaG2-R (CTAAGCTTTTTTCAATTTCTTCAGACTTTGATATATTT). The amplified plasmid DNA was recircularised via In-Fusion cloning, resulting in the transfection construct pDNR-ΔG2/EGFP, in which all but the first 13 amino acids of the g2 coding sequence have been removed. The g2-specific sequence from pDNR-ΔG2/EGFP was introduced into pLP-DHFR/G2 via Cre-loxP site-specific recombination to give the final transfection construct pLP-ΔG2/EGFP. This plasmid was used to introduce a GFP reporter gene into the g2 locus.
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