RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5081
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0102400; Gene model (P.falciparum): PF3D7_0603600; Gene product: AT-rich interactive domain-containing protein, putative
Transgene
Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1319500; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (LAP4; LCCL/lectin adhesive-like protein 4; CCp2)
3'UTR: Gene model: PBANKA_1359600; Gene product: transmission blocking target antigen precursor 6-cysteine protein (P48/45)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: GFP (gfp-mut3)
Promoter: Gene model: PBANKA_0416100; Gene model (P.falciparum): PF3D7_0905300; Gene product: dynein heavy chain, putative
3'UTR: Gene model: PBANKA_1010600; Gene product: calmodulin, putative (cam)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 11 August 2021, 14:57
  *RMgm-5081
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene, Introduction of a transgene
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 P. berghei ANKA 820cl1m1cl1 (RMgm-164)
Other information parent lineP. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517).
The mutant parasite was generated by
Name PI/ResearcherRussell AJC, Billker O
Name Group/DepartmentDepartment of Molecular Biology
Name InstituteUmeå University
CityUmeå
CountrySweden
Name of the mutant parasite
RMgm numberRMgm-5081
Principal namemd4-KO (cl1,2)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteInfertile males, fertile females.
Fertilization and ookineteInfertile males, fertile females.
Complete (or nearly complete) loss in the ability to form oocysts in mosquitoes
OocystComplete (or nearly complete) loss in the ability to form oocysts in mosquitoes
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PBANKA_0102400 and expresses RFP and GFP in female and male gametocytes, respectively.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.08.04.455056

Protein (function)
The gene was selected in a screen for identifying genes essential for for the formation of either male or female gametocytes.
It encodes a conserved Plasmodium protein of unknown function characterized by a putative N-terminal ARID/BRIGHT DNA binding domain, which in other eukaryotes targets developmental transcription factors to AT-rich DNA sequences.

Phenotype
Infertile males, fertile females.
Complete (or nearly complete) loss in the ability to form oocysts in mosquitoes.

Additional information
Ten genes, selected in a screen for identifying genes essential for for the formation of either male or female gametocytes, were targeted for gene deletion in the parent line that produces green-fluorescent males and red-fluorescent females. 

From the paper:
'Flow cytometry analysis of parasites of individual knockout lines confirmed the biased expression of fluorescent sex reporters for all screen hits and further showed sex-specific losses of marker expression. Depending on the affected sex, we refer to the validated genes as “male development” (md1 to md5) or “female development” (fd1 to fd4. As expected, mutants showed a complete or nearly complete loss in their ability to form oocysts in mosquitoes, with the exception of md3, in which oocysts were merely reduced. Genetic crosses using either individual mutants or barcoded single-sex pools showed that for most genes, fertility was sex-specifically affected precisely as predicted by reporter expression. Two notable deviations from the screen results were observed. A cloned mutant in PBANKA_0828000, which by FACS only lacked parasites expressing the female marker, additionally suffered from male infertility; due to its broader gametocyte development (gd) phenotype we refer to this gene as gd1. The second mutant requiring further consideration is md3, for which both a transmission experiment and a cross with a cloned line showed that fertile male gametocytes were still present. However, consistent with the screen result, male fertility of all male mutants, including md3, was reduced to <1% when measured in competition with fertile mutants. We hypothesise that the md3 mutant produces fertile microgametes at a level that is much reduced, but still sufficient to give rise to a relative abundance of oocysts, probably because optimised laboratory infections of P. berghei produce an excess of zygotes, so that oocyst numbers begin to saturate at low input levels.'

For genetic crosses (to analyse male and female fertility) md1, md3, md4, md5, gd1, fd1, fd2, fd3 and fd4 knockout lines were mixed at a 1:1 ratio of iRBC with a nek4 knockout or a hap2 knockout prior to infection with the mixed parasite infected blood intraperitoneally. 

Phenotypes of 10 gene-deletion mutants:
gd1 PBANKA_0828000 - no females, infertile males (PbGEM-091835)

fd1 PBANKA_1454800 - infertile females (N/A)
fd2 PBANKA_0902300 - infertile females (PbGEM-287226)
fd3 PBANKA_1418100 - infertile females (PbGEM-064794)
fd4 PBANKA_1435200 - infertile females (PbGEM-067210)

md1 PBANKA_1302700 - no males, females fertile (N/A)
md2 PBANKA_1447900 - no males, females fertile (PbGEM-340675)
md3 PBANKA_0413400 - few (fertile) males, females fertile (PbGEM-329075)
md4 PBANKA_0102400 - infertile males (PbGEM-327555)
md5 PBANKA_0716500 - infertile males (PbGEM-282066)

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0102400
Gene Model P. falciparum ortholog PF3D7_0603600
Gene productAT-rich interactive domain-containing protein, putative
Gene product: Alternative name
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-327555
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMost gene targeting vectors were obtained from the PlasmoGEM resource (http://plasmogem.sanger.ac.uk/search) and most vectors carried the default PlasmoGEM 3xHA-hdhfr-yfcu gene replacement cassette.
Since PlasmoGEM vectors were not available to target md1 and fd1, knock-out vectors were prepared using PCR.
For md1 amplicons of 1.25 kb upstream and downstream of the gene were amplified from genomic DNA and then assembled either side of a selection cassette amplified from the PlasmoGEM 3xHA-hdhfr-yfcu gateway vector by Gibson Assembly using primer overhangs. The Gibson product was used as the input for a PCR reaction to amplify the entire construct, which was then gel-purified prior to transfection.
For fd1 a CRISPR/Cas9 knock-out vector was constructed by PCR amplification of 0.5 kb 5’ and 3’ regions of the coding sequence of the target gene from genomic DNA, which were restrictionligation cloned so to flank an eef1ɑ 5’UTR-tgdhfr-CAM 3’UTR resistance cassette in a vector also holding the U6 RNA Polymerase 3 promoter from Plasmodium yoelii to drive expression of the target specific guide RNA (gRNA) cloned in by BsmBI, to generate plasmid ABR063. The fd1 CRISPR/Cas9 knock-out line was generated in a split-Cas9 line where conditional activation of CAS9 is achieved by rapamycin-induced dimerisation of a C-terminal fragment of Cas9 fused to FK506- and rapamycin-Binding Protein domain (C-Cas9-FKBP) and a N-terminal fragment of Cas9 fused to the FKBP-Rapamycin Binding domain (N-Cas9-FRB). To achieve this line, N-Cas9-FRB and C-Cas9-FKBP was cloned so to flank the bidirectional 5’UTR of eef1ɑ, and to become nested within 5’ and 3’ targeting sites for the p230p locus generating plasmid ABR010.

For scRNA sequencing experiments, where possible, FACS sortable knockout vectors were generated by converting PlasmoGEM intermediate vectors into knockout vector with a gateway cassette containing an pbhsp70 5’utr-GFPmut3-pbdhfr 3’utr-hdhfr-yfcu expression cassette for constitutive expression of GFP upon vector integration, which allows for FACS sorting to select wild-type free ko parasites without the need for dilution cloning. The gateway construct was generated by amplifying a 1.4 kb fragment of the 5’utr of hsp70 from P. berghei genomic DNA , which was cloned into the R6K-GFPmut3-hdhfr-yfcu Gateway vector (added to the PlasmoGEM tagging vector repertoire), upstream of GFPmut3 to generate the R6K-hsp70p-GFPmut3-hdhfr-yfcu Gateway vector.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameRFP
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1319500
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP4; LCCL/lectin adhesive-like protein 4; CCp2
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_1359600
Gene producttransmission blocking target antigen precursor 6-cysteine protein
Gene product: Alternative nameP48/45
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mut3)
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0416100
Gene Model P. falciparum ortholog PF3D7_0905300
Gene productdynein heavy chain, putative
Gene product: Alternative name
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_1010600
Gene productcalmodulin, putative
Gene product: Alternative namecam
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