RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5584

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_1119200; Gene model (P.falciparum): PF3D7_0620000; Gene product: secreted ookinete protein 25, putative \| ookinete surface-associated protein 8, putative (PSOP25, POS8) Details mutation: The P. berghei psop25 gene replaced by P. vivax psop25 (PVX_114125) and C-terminal tagged with 3xHA
Phenotype	No phenotype has been described

Last modified: 19 November 2024, 18:37

*RMgm-5584

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene mutation
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 38865344
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/Researcher	Zhang B, Zeng Y
Name Group/Department	Department of Immunology, College of Basic Medical Sciences
Name Institute	China Medical University
City	Shenyang, Liaoning
Country	China
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Name of the mutant parasite
RMgm number	RMgm-5584
Principal name	TrPvPSOP25Pb
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Not different from wild type
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the mutant the P. berghei psop25 gene is replaced by P. vivax psop25 gene (PVX_114125) and C-terminal tagged with 3xHA Protein (function) Psop25 encodes a 350 amino acid (aa) protein with a signal peptide, and the native protein is predicted to be 40 kDa. Psop25 transcript is highly expressed in ookinetes. The protein is expressed in ookinetes. The protein is called POS8 in another paper (see RMgm-1299). See also mutant RMgm-4065. Phenotype No discernible differences in parasite asexual growth, the number of macrogametes, and the female/male sex ratio when compared to the WT parasite. As observed previously, ΔPbpsop25 (RMgm-4065) slightly reduced the exflagellation of male gametocytes and substantially reduced the formation of ookinetes. However, introducing Pvpsop25 completely restored the slight male gametocyte exflagellation defect, and the number of ookinetes formed was similar to the WT. These observations demonstrated the conserved function of the PSOP25 proteins in the transmission stages of P. vivax and P. berghei. Additional information PbPSOP25 was predominately expressed on the surface of zygotes, retorts, and mature ookinetes. To characterize the expression of PvPSOP25 in transgenic parasites, IFA was performed with zygotes, retorts, and ookinetes. Both anti-HA monoclonal antibody (mAb) and anti-rPvPSOP25 sera were used to detect the PvPSOP25. A localization pattern consistent with the previous study on PbPSOP25 in WT parasites was observed. The fluorescence signal showed association with the plasma membrane at these stages and co-localized with Pbs25, an ookinete surface marker. Anti-rPvPSOP25 sera did not react with the PbPSOP25 in P. berghei. Other mutants

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1119200

Gene Model P. falciparum ortholog

PF3D7_0620000

Gene product

secreted ookinete protein 25, putative | ookinete surface-associated protein 8, putative

Gene product: Alternative name

PSOP25, POS8

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Details of the genetic modification

Short description of the mutation

The P. berghei psop25 gene replaced by P. vivax psop25 (PVX_114125) and C-terminal tagged with 3xHA

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

To generate the chimeric TrPvPSOP25Pb parasite line, we replaced the Pbpsop25 gene (PBANKA_1119200) with the Pvpsop25 gene (PVX_114125) in P. berghei.
The plasmid pL0034 was used to generate the Pbpsop25 knockout strain (ΔPbpsop25) in our previous study. The transgenic P. berghei expressing full-length Pvpsop25 (without stop codon) in frame with a 3×HA cassette (TrPvPSOP25Pb) was obtained using the double-crossover homologous recombination strategy. The PbPSOP25 5’ and 3’ flanking regions were amplified using primer pairs ΔPbpsop25-5’UTR-F-ΔPbpsop25-5’UTR-R and ΔPbpsop25-3’UTR-F-ΔPbpsop25-3’UTR-R, respectively, and cloned into the vector to flank the human dhfr expression cassette. The full-length Pvpsop25 fragment with 3×ha tag was synthesized by GenScript Biotech Corporation and cloned into the above plasmid at the PstI site adjacent to the PbPSOP25 5’UTR. Plasmid (20 μg) was linearized by ApaI and NotI digestion and electroporated into purified P. berghei schizonts

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

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