RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-5261

Malaria parasite	P. yoelii
Genotype
Disrupted	Gene model (rodent): PY17X_1453700; Gene model (P.falciparum): PF3D7_1236600; Gene product: p25-alpha family protein, putative
Phenotype	Gametocyte/Gamete;

Last modified: 16 November 2022, 16:18

*RMgm-5261

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 36316012
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. yoelii
Parent strain/line	P. y. yoelii 17XNL
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Zhang C, Liu M
Name Group/Department	Department of Microbiology and Parasitology; Anhui Provincial Laboratory of Microbiology and Parasit
Name Institute	School of Basic Medical Sciences, Anhui Medical University
City	Hefei
Country	China
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Name of the mutant parasite
RMgm number	RMgm-5261
Principal name	Pyp25α¯ (KO)
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	Normal production of male and female gametocytes. Reduced exflagellation of male gametocytes
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of PY17X_1453700 Protein (function) The protein contains a domain, called p25α, which is homologous to mammalian tubulin polymerization promoting protein (TPPP/p25), which was originally identified in the brain as a phosphoprotein and has been shown to promote tubulin polymerization of the microtubule (MT). The domain p25α is a part of tubulin polymerization promoting protein (TPPP/p25) family which is conserved among all the ciliated organisms. Phenotype Normal development/growth/multiplication of asexual blood stages in mice. Normal production of male and female gametocytes. Reduced exflagellation of male gametocytes (based on limited analyses) 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

PY17X_1453700

Gene Model P. falciparum ortholog

PF3D7_1236600

Gene product

p25-alpha family protein, putative

Gene product: Alternative name

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(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

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

A 20 bp sequence of guide RNA (gRNA) was designed upstream of the protospacer-adjacent motif (PAM), using the online CRISPR guide RNA/DNA design tool for eukaryotic pathogen (EuPaGDT; http://grna.ctegd.uga.edu), and a pair of complementary oligonucleotides were synthesized for each target site (Sangon Biotech, China). The CRISPR/Cas9 plasmid pYC, a gift from professor Jing Yuan (Xiamen University), was used for all the genetic modifications. Since the PyU6 promoter requires a guanosine nucleotide to initiate transcription, we append an extra “G” base to the beginning of the guide sequence to facilitate U6 transcription initiation if the first base of the guide sequence is not “G”. In addition, the oligonucleotides were designed to generate overhangs to be used for cloning into BsmBI-digested pYC plasmid. The cloned gRNA was placed under the PyU6 promoter and fused with a tracrRNA, which generated a sgRNA. For gene deleting, 5' -genomic and 3' -genomic segments of the target genes were amplified as left and right homologous arms, respectively, using gene-specific primers. The PCR products were digested with using HindIII and AflII restriction enzymes, and then inserted into matched restriction sites of pYC plasmid, which has the human dihydroreductase gene as drug selectable marker. Thus, the transfected parasite with this plasmid can be selected using pyrimethamine

In short: We constructed a plasmid pYC-Py05543 (Pyp25α) containing a 46 bp insert DNA flanked by two homologous regions of Py05543 (403 bp of the 5'-flanking region and 453 bp of the 3'-flanking region) to target the 3'-end of the Py05543 exon 2. One day after electroporation of the plasmid pYC-Py05543 into the P. yoelii 17XNL strain, parasites were selected with pyrimethamine.

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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