RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-907
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0830400; Gene model (P.falciparum): PF3D7_0929600; Gene product: G2 protein, putative (G2)
Transgene
Transgene not Plasmodium: eGFP
Promoter: Gene model: PBANKA_0830400; Gene model (P.falciparum): PF3D7_0929600; Gene product: G2 protein, putative (G2)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_0830400; Gene product: G2 protein (G2)
Phenotype Fertilization and ookinete; Sporozoite; Liver stage;
Last modified: 21 June 2013, 22:09
  *RMgm-907
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
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-907
Principal nameG2-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal numbers of gametes and ookinetes are produced. G2-KO ookinetes show aberrant morphology. G2-KO ookinetes were typically wider and shorter and possessed a bulging area near the central part of the cell. G2-KO parasite-infected mosquitoes formed oocysts that developed normally (although in reduced numbers) and formed large numbers of sporozoites. However, the sporozoites were also of abnormal shape being shorter and possessing an enlarged, bulging area typically near the middle or posterior end of the cell. Sporozoites did not invade salivary glands.
OocystNot different from wild type
SporozoiteNormal numbers of gametes and ookinetes are produced. G2-KO ookinetes show aberrant morphology. G2-KO ookinetes were typically wider and shorter and possessed a bulging area near the central part of the cell. G2-KO parasite-infected mosquitoes formed oocysts that developed normally (although in reduced numbers) and formed large numbers of sporozoites. However, the sporozoites were also of abnormal shape being shorter and possessing an enlarged, bulging area typically near the middle or posterior end of the cell. Sporozoites did not invade salivary glands.
Liver stageSporozoites did not invade salivary glands and transmission to mice by mosquito bite did not occur. Infection of mice by intravenous inoculation of mutant sporozoites has not been reported.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of G2 and expresses GFP under the control of the G2 promoter

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
Phenotype analyses 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).

This morphology phenotype in ookinetes is highly similar to that observed in parasite lines that lack the alveolins IMC1b or IMC1h, and in direct comparison G2-KO ookinetes were indistinguishable from IMC1b-KO or IMC1h-KO ookinetes (RMgm-147, RMgm-601). Null mutants of the alveolins IMC1a, IMC1b and IMC1h display markedly reduced tensile strength. Evidence is presented that the loss of infectivity of the G2 null mutants is unlikely to be caused by a loss of tensile strength.

Additional information
Transcript levels of g2 in P. berghei are over 7.5 times higher in wildtype gametocytes than in gametocytes of null mutants for the DDX6-class RNA helicase DOZI (development of zygote inhibited), which is a strong indicator of translational repression.
Northern analysis of purified asexual blood stages, gametocytes and ookinetes revealed the presence of g2-specific mRNA in ookinetes and gametocytes, with highest levels in the latter, an observation that is fully consistent with the predicted translational repression in the gametocyte.

Expression of the g2 gene product was studied using a transgenic parasite line expressing a full-length, carboxy-terminally GFP-tagged fusion protein from its endogenous promoter (see RMgm-908). These 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. Indeed, immuno-gold labelling of ookinete ultrathin sections saw the large majority of gold particles associated with the pellicle. In young oocysts (4 days), GFP fluorescence had largely disappeared, except for the apical cap structure, and at six days  the majority of oocysts lacked any discernible fluorescence. GFP-based fluorescence was again observed in oocysts from around 8-10 days, 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.

Examination of the ultrastructure of ookinetes by electron microscopy showed the presence of an apparently normal pellicle structure and apical collar in the G2 null mutants. A normal looking pellicle structure was also present in G2 null mutant sporozoites. Wildtype P. berghei sporozoites typically possess 16-17 subpellicular microtubules that are arranged in an asymmetrical manner in which all but one closely spaced microtubules occupy approximately two thirds of the perimeter, while a solitary microtubule is positioned opposite. In contrast, the subpellicular microtubule organisation in G2 null mutant sporozoites was notably different with not one, but four equally spaced microtubules occupying approximately one half of the circumference, and the remaining closely-spaced microtubules in the opposite half. These observations indicate that knockout of G2 affects the organization of the subpellicular microtubules.

Other mutants


  Disrupted: Mutant parasite with a disrupted 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
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
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 1ACGAAGTTATCAGTCGACGGTACCATTTTTGGCTAGATTTTATGACTTA
Additional information primer 1pDNR-G2-F (0.6 kb of the 5′UTR)
Sequence Primer 2ATGAGGGCCCCTAAGCTTATACATAATGAATATTCTTTTTTTTGCC
Additional information primer 2pDNR-G2-R (0.6 kb of the 5′UTR)
Sequence Primer 3TAAACCATTGGTCATAATGTGATGTCTTTCATATGATTCTC
Additional information primer 3pLP-G2-F (0.7 kb of the 3′UTR)
Sequence Primer 4CGGCCGCTCTAGCATGAAAAGCATATGATGTTATGAAGAT
Additional information primer 4pLP-G2-R (0.7 kb of the 3′UTR)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameeGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 mutant lacks expression of G2 and expresses eGFP under the control of the G2 promoter. The egfp gene is inserted into the g2-locus
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0830400
Gene Model P. falciparum ortholog PF3D7_0929600
Gene productG2 protein, putative
Gene product: Alternative nameG2
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0830400
Gene productG2 protein
Gene product: Alternative nameG2
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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