RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4617
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1356600; Gene model (P.falciparum): PF3D7_1343600; Gene product: UDP-N-acetylglucosamine pyrophosphorylase, putative (UAP)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1356600; Gene product: UDP-N-acetylglucosamine pyrophosphorylase, putative (UAP)
Transgene
Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1356600; Gene product: UDP-N-acetylglucosamine pyrophosphorylase, putative (UAP)
Phenotype Asexual bloodstage; Oocyst; Sporozoite; Liver stage;
Last modified: 25 November 2019, 14:06
  *RMgm-4617
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) Reference 1 (PMID number) : 31730853
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherStanway RR, Janse CJ, Heussler VT (1)
Name Group/DepartmentInstitute of Cell Biology
Name InstituteUniversity of Bern
CityBern
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4617
Principal nameΔUAP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGrowth rate is slow - [growth rate 0.81] - [95% CI: 0.74-0.88] - [PlasmoGem version 20170802]
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNo formation of oocysts
SporozoiteNo formation of oocysts; No salivary gland sporozoites
Liver stageSee Additional Information: Evidence for a role during liver stage development
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of UAP and expresses mCherry and GFP under constitutive promoters.

Protein (function)
The gene has been selected based on a predicted role in mosquito- or liver-stage development in a high throughput screen where more than 1300 barcoded P. berghei mutants were followed through the complete life cycle.

Phenotype
No salivary gland sporozoites.
Ookinetes and oocysts were produced in normal numbers  but oocysts gradually disappeared over the course of development and failed to give rise to sporozoites.
See below for a role during liver stage development.

Additional information
The gene has been selected based on a predicted role in mosquito- or liver-stage development in a high throughput screen where more than 1300 barcoded P. berghei mutants (PlasmoGem) were followed through the complete life cycle.
Barcodes were quantified in blood stages (B1), midgut oocysts (MG), salivary gland sporozoites (SG) and in blood stages after intravenous infection of mice with sporozoites (B2).
In the different life cycle transitions, barcode quantification was described as not-reduced, reduced or no power. For this gene:
B1-MG: not reduced
MG-SG: no power
SG-B2: reduced (-8.1)

Cross-fertilization between different mutants in the mosquito midgut limits the power of the screen to reveal gene functions during the subsequent diploid and polyploid stages (i.e. zygotes, ookinetes and oocysts). For instance, knock-out mutants in which only one sex is sterile, can transmit their barcodes to the oocyst by inheritance through the fertile sex. As a result, reductions in barcode abundance for these sex-specific knock-out mutants often did not reach significance at the B1-MG conversion. Known gene functions in the diploid/polyploid ookinete were also generally not recapitulated in the screen, presumably due to heterozygous rescue as a result of cross-fertilization. While these observations highlight the need for future screens to be designed specifically to reveal sexual and mosquito stage phenotypes, they also rationalize how knock-out alleles of genes functioning in fertility or ookinete/oocyst development can be transmitted to salivary gland sporozoites to reveal additional gene functions after sporozoite transmission to the vertebrate host.

Liver stage phenotype:
In the paper a method is described where they cross-fertilize wild type parasites with a mutant lacking UAP. The crossing rescues the knock-out phenotype in the mosquito (see above for explanation). Since the knock-out mutant expresses fluorescent markers and a pyrimethamine resistance marker it is possible to analyse the fluorescent haploid knock-out sporozoites during development in the liver and to determine the prepatent period of the knock-out mutant. It was found that mice intravenously infected with defined numbers of mutant sporozoites did not develop blood infections or with a prolonged prepatent period (8.4 days; 5-11).

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1356600
Gene Model P. falciparum ortholog PF3D7_1343600
Gene productUDP-N-acetylglucosamine pyrophosphorylase, putative
Gene product: Alternative nameUAP
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe single knockout parasite line ΔUAP (PBANKA_1356600), was generated using a vector with a “GOMO-GFP-Cherry-FACS” selection cassette, which contains not only the hdhfr::yfcu selectable marker (SM), under the control of the P. berghei eef1α promoter region and 3’ terminal sequence of pbdhfr but additionally cassettes for GFP and mCherry expression, controlled by the hsp70 and eef1α promoters respectively and terminated by the 3’ terminal sequence of pbdhfr. The GOMO cassette was adapted for use with PlasmoGEM vectors in collaboration with Olivier Silvie and adapted from (Manzoni et al., 2015).
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 nameGFP
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) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_1356600
Gene productUDP-N-acetylglucosamine pyrophosphorylase, putative
Gene product: Alternative nameUAP
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 namemCherry
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) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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 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_1356600
Gene productUDP-N-acetylglucosamine pyrophosphorylase, putative
Gene product: Alternative nameUAP
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