RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5271

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_0305900; Gene model (P.falciparum): PF3D7_0208800; Gene product: protein P22, putative (pb22) Details mutation: The P. berghei Pb22 gene replaced by the P. vivax Pv22 gene
Phenotype	No phenotype has been described

Last modified: 29 December 2022, 15:24

*RMgm-5271

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) : 36503858
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	RMgm-4923
Other information parent line	In mutant RMgm-4923 the Pb22 gene (open reading frame) is deleted by introducing the hdhfr/yfcu selectable marker cassette
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The mutant parasite was generated by
Name PI/Researcher	Bai J, Cao Y
Name Group/Department	Department of Immunology, College of Basic Medical Sciences
Name Institute	China Medical University
City	Shenyang
Country	China
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Name of the mutant parasite
RMgm number	RMgm-5271
Principal name	TrPv22Pb
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 different from wild type
Sporozoite	Not different from wild type
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation In the mutant the Pb22 gene of P. berghei has been replaced by the Pv22 gene of P. vivax. The P. vixax Pv22 is introduced in mutant RMgm-4923 in which the Pb22 gene (open reading frame) has been deleted by introducing the hdhfr/yfcu selectable marker cassette. The TrPv22Pb mutant has been selected by negative selection. Protein (function) PBANKA_0305900 (Pb22), which is predicted to encode a sexual-stage protein of 218 amino acids (aa) with a molecular weight of 22 kDa. Pb22 has a putative signal peptide (aa 1–18), suggesting that it might be secreted. Pb22 did not contain any domains of known functions. Analysis of P. berghei mutants lacking Pb22 (RMgm-4923) showed defects in formation of male gametes (exflagellation), reduced ookinete formation and absence of oocyst formation. Phenotype Analysis of P. berghei mutants lacking Pb22 (RMgm-4923) showed defects in formation of male gametes (exflagellation), reduced ookinete formation and absence of oocyst formation. Analysis of the mutant expressing P. vivax Pv22 showed a normal/wildtype phenotype throughout the complete lifecycle, indicating complementation of the function of Pb22 by Pv22. Additional information Pv22 expression was low in early asexual stages but similarly high in schizonts, gametocytes, and ookinetes. Pv22 was expressed in asexual blood stages, localized in the cytosol ring and trophozoite stages, but in schizonts it appeared to be associated with the merozoite surface . In sexual stages, Pv22 was detected in both male and female gametocytes, gametes and ookinetes. Of particular relevance is the peripheral localization of Pv22 in gametes, zygotes and ookinetes. From the Abstract of the paper: Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22- immunized mice infected with TrPv22Pb parasites showed a 49.3–53.3 % reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. Other mutants

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0305900

Gene Model P. falciparum ortholog

PF3D7_0208800

Gene product

protein P22, putative

Gene product: Alternative name

pb22

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Details of the genetic modification

Short description of the mutation

The P. berghei Pb22 gene replaced by the P. vivax Pv22 gene

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/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

Selection (negative) procedure

5-fluorocytosine (5-FC)

Additional remarks genetic modification

In the mutant the Pb22 gene of P. berghei has been replaced by the Pv22 gene of P. vivax.
The P. vixax Pv22 is introduced in mutant RMgm-4923 in which the Pb22 gene (open reading frame) has been deleted by introducing the hdhfr/yfcu selectable marker cassette. The TrPv22Pb mutant has been selected by negative selection.

To generate a transgenic P. berghei expressing Pv22 with a 3xHA tag referred to as TrPv22Pb, the complete pv22 open reading frame flanked by the 5' and 3' UTR of pb22 was inserted into the pUC57 vector at the HindIII and NotI sites. The plasmid was linearized and electroporated into purified schizonts of mutant RMgm-4923.

Additional remarks selection procedure

The P. vixax Pv22 is introduced in mutant RMgm-4923 in which the Pb22 gene (open reading frame) has been deleted by introducing the hdhfr/yfcu selectable marker cassette. The TrPv22Pb mutant has been selected by negative selection.

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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