RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5588

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1122300; Gene model (P.falciparum): PF3D7_0623400; Gene product: MEI2-like RNA-binding protein (Mei2)
Phenotype	Liver stage;

Last modified: 9 December 2024, 10:01

*RMgm-5588

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption
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	Le S, Beattie L
Name Group/Department	The Peter Doherty Institute for Infection and Immunity
Name Institute	University of Melbourne
City	Parkville
Country	Australia
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Name of the mutant parasite
RMgm number	RMgm-5588
Principal name	PbΔmei2
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	The phenotype has not been analysed/described in detail in the paper. See for detailed analyses RMgm-4937 for the phenotype of a mutant lacking expression of MEI2: Normal blood stage and mosquito stage development. Normal infectivity of sporozoites to hepatocytes in vitro and in vivo. Mutant parasites lacking expression of MEI 2 develop into large, mature liver stages with extensive nuclear division and expression of merozoite specific proteins, such as msp1 and ama1. Most of the parasites arrest growth, however, just before the formation of infective merozoites. Only after infection of mice with high doses of sporozoites (2x10(5)) some of the mice (3 out of 10) developed blood infections with a greatly prolonged prepatent period (12-14 days).
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of MEI2 Published in bioRxiv preprint doi: https://doi.org/10.1101/2024.12.06.627130 Protein (function) Mei2 is a member of the largest family of RNA binding proteins (RBPs) – those that contain a RNA recognition motif (RRM), a stretch of 70-90 amino acids that contain two consensus RNA-interacting motifs, RNP1 and RNP2. RRM-containing proteins are subdivided into ten separate families (RRM_1 thru RRM_10) based on shared amino acid identities between members of each family and Mei2 contains a C-terminal RRM_2, thought to be unique to fungi and plants. Plasmodium contains a single Mei2-like gene. See for detailed analyses RMgm-4937 for the phenotype of a mutant lacking expression of MEI2: Normal blood stage and mosquito stage development. Normal infectivity of sporozoites to hepatocytes in vitro and in vivo. Mutant parasites lacking expression of MEI 2 develop into large, mature liver stages with extensive nuclear division and expression of merozoite specific proteins, such as msp1 and ama1. Most of the parasites arrest growth, however, just before the formation of infective merozoites. Only after infection of mice with high doses of sporozoites (2x10(5)) some of the mice (3 out of 10) developed blood infections with a greatly prolonged prepatent period (12-14 days). Phenotype The phenotype has not been analysed/described in detail in the paper. See for detailed analyses RMgm-4937 for the phenotype of a mutant lacking expression of MEI2: Normal blood stage and mosquito stage development. Normal infectivity of sporozoites to hepatocytes in vitro and in vivo. Mutant parasites lacking expression of MEI 2 develop into large, mature liver stages with extensive nuclear division and expression of merozoite specific proteins, such as msp1 and ama1. Most of the parasites arrest growth, however, just before the formation of infective merozoites. Only after infection of mice with high doses of sporozoites (2x10(5)) some of the mice (3 out of 10) developed blood infections with a greatly prolonged prepatent period (12-14 days). Additional information Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1122300

Gene Model P. falciparum ortholog

PF3D7_0623400

Gene product

MEI2-like RNA-binding protein

Gene product: Alternative name

Mei2

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

CRISPR/Cas9 construct: integration through double strand break repair

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

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

Generation of PbDmei2 parasites PbDmei2 parasites were generated. The following was performed with the gene region for mei2 reversed so that the coding sequence reads left to right. 5’ and 3’ homology flanks were amplified from Pb genomic DNA with the primers CJ472 + CJ473, and CJ474 + CJ475 511, producing amplicons of 798bp and 832bp respectively. The 5’ flank reverse primer CJ473 and the 3’ flank forward primer CJ474 both featured a 23 base multi-cloning-site complementary tail (including a possible guide sequence), which was utilized in a further PCR reaction to generate the 5’, 3’ flank fusion fragment. This was cloned into the CRISPR/Cas9 plasmid pYC_L2 (a kind gift from Ashley Vaughan) with the restriction enzymes KpnI and EcoRI. Two guide sequences (Guide 3 and 28) were identified with CHOPCHOP66 and each sequence cloned into separate KO flank containing plasmids with the enzyme Esp3I to generate the mei2KO plasmid pCJ133 and 134 respectively. 15ug of each plasmid was co-tranfected into into magnet-purified Pb schizont stage parasites and injected intravenously into Swiss Webster mice. After 24 hours, mice were put on water containing 70 μg/mL pyrimethamine. Clonal parasites generated by limiting dilution

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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