RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4992

Malaria parasite	P. yoelii
Genotype
Transgene	Transgene not Plasmodium: mCherry Promoter: Gene model: PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP) 3'UTR: Gene model: PBANKA_0403200; Gene product: circumsporozoite (CS) protein (CSP) Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230p)
Phenotype	Sporozoite;

Last modified: 17 May 2021, 08:55

*RMgm-4992

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Introduction of a transgene
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 33750026
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	RMgm-688
Other information parent line	GIMO-Py17X (RMgm-688; line 1923cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PY04774) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. yoelii 17XNL line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/Researcher	Hopp CS, Sinnis P
Name Group/Department	Department of Medicine, Division of Infectious Diseases
Name Institute	Johns Hopkins University School of Medicine
City	Baltimore
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-4992
Principal name	PymCherry
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	mCherry expression in sporozoites
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses mCherry under control of the csp regulatory sequences (3'and 5'-UTR). The mCherry expression cassette has been introduced into the neutral 230p locus (by GIMO transfection and negative selection). Protein (function) Phenotype mCherry expression in sporozoites. Additional information Other mutants

Transgene: Mutant parasite expressing a transgene

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Type and details of transgene

Is the transgene Plasmodium derived

Transgene: not Plasmodium

Transgene name

mCherry

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Details of the genetic modification

Inducable system used

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

hdhfr/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

Selection (negative) procedure

5-fluorocytosine (5-FC)

Additional remarks genetic modification

The targeting plasmid pL1849‐mCherryCSP, containing sequences that would target the mcherry transgene to the P. yoelii 230p locus, and the mcherry coding sequence flanked by the P. berghei csp 5’UTR and the P. berghei dhfr 3’UTR was generated as follows: the 5’pbcsp‐mCherry‐3ʹpbdhfr cassette was amplified from plasmid pL0047 (RMgm database http://www.pberghei.eu), with primers pL0047‐PbCS‐mCherry‐F and pL0047‐PbCS‐mCherry‐R, which added XmaI sites for cloning into pL1849 (Lin et al, 2011), which contained the P. yoelii 230p locus (PY03857) 5'‐ and 3'‐targeting sequences. The resulting construct pL1849‐mCherryCSP was released from the plasmid through digestion with BstXI and ScaI and used for transfection into the P. yoelii line Py17XGIMO (line 1923cl1; Lin et al, 2011), according to standard procedures . The following day, negative selection with 1 mg/ml 5‐FC in drinking water was used to enrich for parasites that integrated the PbCSP‐mCherry‐3ʹpbdhfr cassette and excised the hdhfr::yfcu locus through double cross‐over homologous recombination.

Additional remarks selection procedure

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Other details transgene

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Promoter

Gene Model of Parasite

PBANKA_0403200

Gene Model P. falciparum ortholog

PF3D7_0304600

Gene product

circumsporozoite (CS) protein

Gene product: Alternative name

CSP

Primer information details of the primers used for amplification of the promoter sequence Click to view information

Primer information details of the primers used for amplification of the promoter sequence Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

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3'-UTR

Gene Model of Parasite

PBANKA_0403200

Gene product

circumsporozoite (CS) protein

Gene product: Alternative name

CSP

Primer information details of the primers used for amplification the 3'-UTR sequences Click to view information

Primer information details of the primers used for amplification the 3'-UTR sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

Insertion/Replacement locus

Replacement / Insertion

Replacement locus

Gene Model of Parasite

PY17X_0306600

Gene product

6-cysteine protein

Gene product: Alternative name

P230p; 230p

Primer information details of the primers used for amplification of the target sequences Click to view information

Primer information details of the 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

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