RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-5405

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0605900; Gene model (P.falciparum): PF3D7_1207400; Gene product: conserved Plasmodium protein, unknown function (S14) Name tag: 3xHA-mCherry
Phenotype	Sporozoite;

Last modified: 10 March 2024, 19:38

*RMgm-5405

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Not published (yet)
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Ghosh A, Mishra S
Name Group/Department	Division of Molecular Microbiology and Immunology
Name Institute	CSIR-Central Drug Research Institute
City	Lucknow
Country	India
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Name of the mutant parasite
RMgm number	RMgm-5405
Principal name	S14-3XHA-mCherry
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 tested
Oocyst	Not tested
Sporozoite	S14 expression was observed in sporozoites but not in the blood and liver stages. Analysis of the mCherry pattern on sporozoites revealed localization on the membrane of the sporozoites IFA with anti-CSP and anti-mCherry antibodies confirmed S14 localization on the membrane.
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation The mutant expresses a 3xHA-mCherry tagged version of S14. Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.08.30.555469 Protein (function) The gene encoding S14 is highly transcribed in sporozoites. the protein does not contain a transmembrane domain or signal peptide. Analysis of a mutant lacking S14 expression (RMgm-5404) showed the following: Evidence for normal development inside oocysts and egress of sporozoites in the hemolymph. The number of salivary gland sporozoites strongly reduced. Sporozoites are non-motile (no gliding motility). Sporozoites are unable to infect mice (by mosquito bite or intravenous injection) and unable to invade hepatocytes in vitro. Phenotype S14 expression was observed in sporozoites but not in the blood and liver stages. Analysis of the mCherry pattern on sporozoites revealed localization on the membrane of the sporozoites IFA with anti-CSP and anti-mCherry antibodies confirmed S14 localization on the membrane. Analysis of a mutant lacking S14 expression (RMgm-5404) showed the following: Evidence for normal development inside oocysts and egress of sporozoites in the hemolymph. The number of salivary gland sporozoites strongly reduced. Sporozoites are non-motile (no gliding motility). Sporozoites are unable to infect mice (by mosquito bite or intravenous injection) and unable to invade hepatocytes in vitro. Additional information Evidence is presented that: - S14 localizes on the inner membrane and is essential for parasite gliding motility - S14 associates with the glidosome-associated proteins MTIP (PBANKA_1459500) and GAP45 (PBANKA_1437600) - S14 performs gliding-specific function and absence of S14 does not affect the organization (location/expression) of IMC and surface proteins (MTIP, GAP45, CSP, TRAP). Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0605900

Gene Model P. falciparum ortholog

PF3D7_1207400

Gene product

conserved Plasmodium protein, unknown function

Gene product: Alternative name

S14

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Details of the genetic modification

Name of the tag

3xHA-mCherry

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

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

For the endogenous tagging of S14 with 3XHA-mCherry, two fragments, F1 (0.74 kb) and F2 (0.65 kb), were amplified using 1010/1011 and 1005/1006 and cloned into plasmid pBC-3XHA-mCherry-hDHFR at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI

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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