RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
DisruptedGene model (rodent): PY17X_1453700; Gene model (P.falciparum): PF3D7_1236600; Gene product: p25-alpha family protein, putative
Phenotype Gametocyte/Gamete;
Last modified: 16 November 2022, 16:18
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36316012
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherZhang C, Liu M
Name Group/DepartmentDepartment of Microbiology and Parasitology; Anhui Provincial Laboratory of Microbiology and Parasit
Name InstituteSchool of Basic Medical Sciences, Anhui Medical University
Name of the mutant parasite
RMgm numberRMgm-5261
Principal namePyp25α¯ (KO)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of male and female gametocytes. Reduced exflagellation of male gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of PY17X_1453700

Protein (function)
The protein contains a domain, called p25α, which is homologous to mammalian tubulin polymerization promoting protein (TPPP/p25), which was originally identified in the brain as a phosphoprotein and has been shown to promote tubulin polymerization of the microtubule (MT). The domain p25α is a part of tubulin polymerization promoting protein (TPPP/p25) family which is conserved among all the ciliated organisms.

Normal development/growth/multiplication of asexual blood stages in mice. Normal production of male and female gametocytes. Reduced exflagellation of male gametocytes (based on limited analyses)

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1453700
Gene Model P. falciparum ortholog PF3D7_1236600
Gene productp25-alpha family 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 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 modificationA 20 bp sequence of guide RNA (gRNA) was designed upstream of the protospacer-adjacent motif (PAM), using the online CRISPR guide RNA/DNA design tool for eukaryotic pathogen (EuPaGDT; http://grna.ctegd.uga.edu), and a pair of complementary oligonucleotides were synthesized for each target site (Sangon Biotech, China). The CRISPR/Cas9 plasmid pYC, a gift from professor Jing Yuan (Xiamen University), was used for all the genetic modifications. Since the PyU6 promoter requires a guanosine nucleotide to initiate transcription, we append an extra “G” base to the beginning of the guide sequence to facilitate U6 transcription initiation if the first base of the guide sequence is not “G”. In addition, the oligonucleotides were designed to generate overhangs to be used for cloning into BsmBI-digested pYC plasmid. The cloned gRNA was placed under the PyU6 promoter and fused with a tracrRNA, which generated a sgRNA. For gene deleting, 5' -genomic and 3' -genomic segments of the target genes were amplified as left and right homologous arms, respectively, using gene-specific primers. The PCR products were digested with using HindIII and AflII restriction enzymes, and then inserted into matched restriction sites of pYC plasmid, which has the human dihydroreductase gene as drug selectable marker. Thus, the transfected parasite with this plasmid can be selected using pyrimethamine

In short: We constructed a plasmid pYC-Py05543 (Pyp25α) containing a 46 bp insert DNA flanked by two homologous regions of Py05543 (403 bp of the 5'-flanking region and 453 bp of the 3'-flanking region) to target the 3'-end of the Py05543 exon 2. One day after electroporation of the plasmid pYC-Py05543 into the P. yoelii 17XNL strain, parasites were selected with pyrimethamine.
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