RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5041

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_1219500; Gene model (P.falciparum): PF3D7_0709000; Gene product: chloroquine resistance transporter (CRT) Details mutation: a mutated CRT with amino acid change N331I
Phenotype	Asexual bloodstage;

Last modified: 23 July 2021, 14:44

*RMgm-5041

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) : 34222045
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	Ikeda M, Mita T
Name Group/Department	Department of Tropical Medicine and Parasitology, Faculty of Medicine
Name Institute	Juntendo University
City	Tokyo
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-5041
Principal name	PbCRT-N331I
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Slower growth rate of asexual blood stages and reduced sensitivity to Piperaquine.
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a mutated version of CRT with amino acid change N331I Protein (function) The Chloroquine Resistance Transporter (CRT) is an integral membrane protein localized to the parasite's internal digestive vacuole membrane. Mutations in CRT result in a decreased accumulation of chloroquine within the digestive vacuole in the parasite as a result of increased transport of chloroquine. Phenotype Slower growth rate of asexual blood stages and reduced sensitivity to Piperaquine. Additional information Other mutants

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1219500

Gene Model P. falciparum ortholog

PF3D7_0709000

Gene product

chloroquine resistance transporter

Gene product: Alternative name

CRT

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Details of the genetic modification

Short description of the mutation

a mutated CRT with amino acid change N331I

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

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

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

The plasmid pYC for the CRISPR/Cas9 gene editing of P. yoelii was kindly gifted by Dr. Yuan (Zhang et al., 2014). To customize pYC for P. berghei, the U6 promoter of P. yoelii in pYC was excluded by inverse PCR using the primers A and B, which are designed in an outward direction from the P. yoelii U6 promoter region. The U6 promoter of P. berghei (PBANKA_1354380) was amplified with the primers C and D and ligated into the pYC PCR product by using an In-Fusion HD Cloning kit (TAKARA). The resultant plasmid, pBC, was used for P. berghei genome editing.
As donor DNA, a 1,343 bp fragment containing PbCRT(N331I) was synthesized (Genewiz, Japan) and ligated to the HindIII/AflII site in pBC by an In-Fusion HD Cloning kit. The candidate guide RNA (gRNA) was designed by the Eukaryotic Pathogen CRISPR Guide RNA Design Tool (http://grna.ctegd.uga.edu/). A double-stranded DNA coding for the candidate gRNA was ligated into the BsmBI site of pBC; therefore, the gRNA was transcribed under the P. berghei U6 promoter. The resultant plasmid, pPbCRT(N331I), was electroporated into wild-type P. berghei. Transfection and subsequent parasite cloning followed a previously described protocol (Janse et al., 2006).

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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