RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1355
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1233200; Gene model (P.falciparum): PF3D7_0518400; Gene product: cyclin, putative (P-Type Cyclin CYC3; CYC3)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 5 April 2016, 09:56
  *RMgm-1355
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) : 26565797
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 507cl1 (RMgm-7)
Other information parent lineP. berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherRoques M; Tewari R
Name Group/DepartmentSchool of Life Sciences, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1355
Principal nameΔcyc3
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 ookineteNot different from wild type
OocystNo significant reduction in the number of Δcyc3 oocysts compared to WT controls at 5 dpi. However a significant reduction was observed at 7 dpi, which became even more evident at 10, 14 and 21 dpi. Δcyc3 oocysts were substantially smaller than WT. The number of Δcyc3 sporozoites was significantly reduced compared to WT.
Many (>60%) of the Δcyc3 oocysts showed no evidence of sporulation
SporozoiteNo significant reduction in the number of Δcyc3 oocysts compared to WT controls at 5 dpi. However a significant reduction was observed at 7 dpi, which became even more evident at 10, 14 and 21 dpi. Δcyc3 oocysts were substantially smaller than WT. The number of Δcyc3 sporozoites was significantly reduced compared to WT.
Sporozoites, however, showed a normal infectivity to mice.
Liver stageThe number of Δcyc3 sporozoites was significantly reduced compared to WT.
Sporozoites, however, showed a normal infectivity to mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CYC3 and expresses GFP under the constitutive eef1a promoter

Protein (function)
Plasmodium species encode only three identifiable cyclins–CYC1, CYC3 and CYC4. A P. falciparum gene (encoded by PF3D7_1227500) annotated as PfCYC2 did not show a significant match to any of the cyclin-specific HMMs built during these analyses (even at extremely liberal thresholds), nor to the domains built by PFam. This protein was originally identified as a cyclin on the basis of a very limited similarity to a cyclin A from the sea star Patiria (Asterina) pectinifera (13% identity across alignable length), but it lacks key alignable residues across most of the cyclin box and has no detectable cyclin-like function in biochemical assays. These data strongly suggest that CYC2 is not a true cyclin.

The Plasmodium cyclin repertoire is highly unusual in that it entirely lacks Group I, the largest group of cyclins. This group contains most canonical cyclin families that regulate specific cell cycle transitions with their CDK partners: Cyclin D-CDK4/6 for G1 progression, Cyclin E-CDK2 for the G1/S transition, Cyclin A-CDK2 for S phase progression and CyclinA/B-CDK1 for mitosis, although in fission yeast, all cell cycle transitions are driven by a single Cyclin B/CDK complex (CDC13/CDC2). In keeping with this key role and previous analyses, Group I cyclins were found in all non-apicomplexan species examined, including the alveolate Tetrahymena thermophila. However, none of the apicomplexan species examined contained Group I cyclins, except Cryptosporidium, which was found to encode three Group I cyclins of indeterminate family, suggesting that there has been a loss of Group I cyclins during the evolution of apicomplexan lineages. Plasmodium species encode only one cyclin, CYC3, from the P family (Group II) and two Group III cyclins from families H and L. The cyclin P family is not found in animals, but includes many plant cyclins and Pho80 in budding yeast, which link nutritional sensing to cell cycle progression. In contrast, both H and L families are associated with transcription: the CDK7/Cyclin H/MAT1 complex functions as a Cdk-activating kinase in cell cycle regulation and as a modulator of the general transcription factor TFIIH. Similarly, CDK11-Cyclin L complex in fission yeast regulates the formation of the Mediator complex, a coactivator of RNA polymerase II transcription. 

Phenotype
Reduced oocyst and sporozoite formation. Aberrant oocyst formation

Additional information
Quantitative RT-PCR (qRT-PCR) was used to investigate the RNA levels of cyc1, 3 and 4 in six stages of the wild-type parasite life cycle. The transcription profiles showed expression of the cyclins throughout parasite development with the highest RNA levels for all three cyclins found in gametocytes (both non-activated and activated) and schizonts (particularly cyc4). The cyc3 RNA level was highest in non-activated gametocytes, whereas for cyc1 and cyc4, the levels were highest in activated gametocytes.

See RMgm-1356 for a mutant expressing a C-terminal GFP-tagged version of CYC3.
CYC3-GFP parasites were able to complete the full life cycle with no detectable phenotype observed from tagging with GFP including oocyst development at 14 days post infection (dpi) in mosquitoes. Live imaging of parasites revealed CYC3-GFP presence throughout the parasite cell body with a predominantly cytosolic localisation at most of the key Plasmodium life cycle stages examined (trophozoite, male and female gametocyte, zygote, ookinete, oocyst and salivary gland sporozoite). Deconvolution fluorescence imaging showed that CYC3-GFP was uniformly present throughout both the cytoplasm and the nucleus in trophozoites, gametocytes and ookinetes and noticeably enriched in the nucleus of ookinetes.
Fluorescent imaging showed no detectable CYC3-GFP expression in oocysts at 5 and 7 dpi. Expression of CYC3-GFP was first observed at low levels in the majority of oocysts at 10 dpi with the highest expression detected at 14 dpi. Oocysts that had formed fully mature sporozoites showed the highest protein expression. After 14 dpi, a decrease was observed in CYC3-GFP expression in oocysts up to day 21pi.

Other mutants
See RMgm-1356 for a mutant expressing a C-terminal GFP-tagged version of CYC3
See RMgm-1301 for an independent mutant lacking expression of CYC3


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1233200
Gene Model P. falciparum ortholog PF3D7_0518400
Gene productcyclin, putative
Gene product: Alternative nameP-Type Cyclin CYC3; CYC3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
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 modificationPCR primers N0451 and N0452 were used to generate a 411 bp fragment of 5' upstream sequence of cyc3 from genomic DNA, which was inserted into ApaI and HindIII restriction sites upstream of the dhfr/ts cassette of pBS-DHFR. A 663 bp fragment generated with primers N0453 and N0454 from the 3' flanking region of cyc3 was then inserted downstream of the dhfr/ts cassette using EcoRI and XbaI restriction sites. The linear targeting sequence was released using ApaI/XbaI.
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 1CCCCGGGCCCGCTTTATATTTCAGTTTATAGTGTCTTTC
Additional information primer 1N0451
Sequence Primer 2GGGGAAGCTTGTAAAACAAAACATTCATTACTACAACC
Additional information primer 2N0452
Sequence Primer 3CCCCGAATTCGCAAACCAATTTCAGTCGTTAAAACTTATAACC
Additional information primer 3N0453
Sequence Primer 4GGGGTCTAGAGCATTATTATATTTTCCTAACTTTTG
Additional information primer 4N0454
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 nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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 PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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