RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0306700; Gene model (P.falciparum): PF3D7_0209600; Gene product: transporter, putative (signaling linking factor (SLF), putative)
PhenotypeNo phenotype has been described
Last modified: 18 February 2024, 18:34
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37327340
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherKuehnel RM, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0306700
Gene Model P. falciparum ortholog PF3D7_0209600
Gene producttransporter, putative
Gene product: Alternative namesignaling linking factor (SLF), putative
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene deletion–targeting vector for pdeα was constructed using the pAB022 plasmid, which contains polylinker sites flanking an hdhfr expression cassette conferring resistance to pyrimethamine. Polymerase chain reaction (PCR) primers MB763 and MB764 were used to generate a fragment of pdeα 5′ upstream sequence from genomic DNA, which was inserted into Hind III and Pst I restriction sites upstream of the hdhfr cassette. A fragment generated with primers MB765 and MB766 from the 3′ flanking region of pdeα was then inserted downstream of the hdhfr cassette using Kpn I and Not I restriction sites. The linear targeting sequence was released using Hind III and Not I, and the construct was transfected into the ANKA line 2.34 or PDEδ-KO.

3xHA, KO, or AID/HA tagging of GCα, UGO, and PDEγ was generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of KO and tagging constructs was performed using sequential recombineering and gateway steps as previously described. For each gene of interest (goi), the Zeocin resistance/Phe sensitivity cassette was introduced using oligonucleotides goi HA-F × goi HA-R and goi KO-F × goi KO-R for 3xHA, AID/HA tagging, and KO targeting vectors. Insertion of the gateway (GW) cassette following gateway reaction was confirmed using primer pairs GW1 × goi QCR1 and GW2 × goi QCR2. The modified library inserts were then released from the plasmid backbone using Not I. The UGO-AID/HA and GCα-AID/HA targeting vectors were transfected into the 615 parasite line, and the PDEγ-KO, UGO-3xHA, and GCα-3xHA vectors into the 2.34 line
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