RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5224

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_1450200; Gene model (P.falciparum): PF3D7_1235600; Gene product: Serine hydroxymethyltransferase, putative (SHMT) Details mutation: The P. berghei shmt gene replaced with the P. vivax shmt gene (PVX_100730)
Phenotype	Asexual bloodstage;

Last modified: 10 August 2022, 12:12

*RMgm-5224

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) : 23173711
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	Pornthanakasem W, Leartsakulpanich U
Name Group/Department	National Center for Genetic Engineering and Biotechnology
Name Institute	National Center for Genetic Engineering and Biotechnology
City	Khlong Luang, Pathum Thani
Country	Thailand
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Name of the mutant parasite
RMgm number	RMgm-5224
Principal name	ΔPbPvcshmt
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	(Slight) reduction in asexual blood stage growth/multiplication in mice
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the mutant the P. berghei shmt gene has been replaced by the P. vivax shmt gene ortholog (PVX_100730) Protein (function) Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT). Plasmodium falciparum contains PF3D7_1235600, a previously characterized cSHMT gene (Pfcshmt), and PF3D7_1456100, a putative gene of mSHMT (Pfmshmt). Phenotype The generation of mutant ΔPbPvcshmt indicates that P. vivax SHMT can (partially) complement P. berghei (SHMT) in blood stages since knock-out of the P. berghei shmt gene was not possible (see RMgm-5223). However, mutant parasites showed a (slight) reduction in asexual blood stage growth/multiplication in mice. 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_1450200

Gene Model P. falciparum ortholog

PF3D7_1235600

Gene product

Serine hydroxymethyltransferase, putative

Gene product: Alternative name

SHMT

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Details of the genetic modification

Short description of the mutation

The P. berghei shmt gene replaced with the P. vivax shmt gene (PVX_100730)

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

tgdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Plasmids for the study of gene knockout in Plasmodium berghei ANKA strain were constructed based on the sequence of pL0017 vector (The Malaria Research and Reference Reagent Resource Center; MR4), which contains Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr/ts) and green fluorescent protein gene (gfp) expression cassettes for pyrimethamine (PYR) selection and fluorescence detection of transfected parasites. The 553 and 1,018 bp of PCR amplicons, corresponding to 5′- and 3′UTR of Pbcshmt (PBANKA_145020) respectively, were produced initially from P. berghei genomic DNA (gDNA). The 5′UTR fragment was inserted into pL0017 at Hind III site, while the 3′UTR fragment was inserted at Kpn I and Sac II sites respectively. This construct, pL0017_Δshmt, was used in the knockout study. For allelic replacement construct, gfp in pL0017_Δshmt was replaced with Plasmodium vivax cshmt (Pvcshmt; PVX_100730) and named pL0017_(Pv)Δshmt.

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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