RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1024

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein \| sporozoite surface protein 2 (TRAP; SSP2; SSP-2) Name tag: triple myc
Phenotype	Sporozoite;

Last modified: 13 April 2014, 16:40

*RMgm-1024

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 24617597
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	RMgm-683
Other information parent line	The mutant expresses the yeast FlpL recombinase under the control of the promoter of trap. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome.
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The mutant parasite was generated by
Name PI/Researcher	Carey, AF; Amino, R
Name Group/Department	Unité de Biologie et Genétique du Paludisme
Name Institute	Institut Pasteur
City	Paris
Country	France
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Name of the mutant parasite
RMgm number	RMgm-1024
Principal name	TRAP-Myc
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	This parasite expresses 3xcmyc-tagged TRAP in sporozoites
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation The mutant expresses C-terminal 3xcmyc-tagged TRAP. Protein (function) Phenotype This parasite expresses 3xcmyc-tagged TRAP in sporozoites Additional information The TRAP-Myc parasite was generated by cloning the TRAP open reading frame and inserting a triple Myc tag into the disorganized region of the TRAP ectodomain, after base pair 1482. This was flanked upstream by 1.3kB of the TRAP 5’ region, and downstream by an F3 site and 1.2 kB of the TRAP 3’ region, and cloned into a vector which contains the hDHFR resistance cassette flanked by FRT sites. This construct was integrated by double crossover replacing the endogenous TRAP, using as homology regions the TRAP 3' and the pUC18 plasmid backbone, into the TRAP/FlpL deleter strain, a P. berghei NK65 GFP fluorescent strain (RMgm-683) in which the Flip recombinase was integrated at the TRAP locus and had the marker recycled, leaving as integration "scar" a pUC18 plasmid backbone upstream of TRAP. The hDHFR resistance cassette was then excised by cycling through the mosquito stage Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1349800

Gene Model P. falciparum ortholog

PF3D7_1335900

Gene product

thrombospondin-related anonymous protein | sporozoite surface protein 2

Gene product: Alternative name

TRAP; SSP2; SSP-2

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Details of the genetic modification

Name of the tag

triple myc

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

PCR construct 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

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The TRAP-Myc parasite was generated by cloning the TRAP open reading frame and inserting a triple Myc tag into the disorganized region of the TRAP ectodomain, after base pair 1482. This was flanked upstream by 1.3kB of the TRAP 5’ region, and downstream by an F3 site and 1.2 kB of the TRAP 3’ region, and cloned into a vector which contains the hDHFR resistance cassette flanked by FRT sites. This construct was integrated by double crossover replacing the endogenous TRAP, using as homology regions the TRAP 3' and the pUC18 plasmid backbone, into the TRAP/FlpL deleter strain, a P. berghei NK65 GFP fluorescent strain (RMgm-683) in which the Flip recombinase was integrated at the TRAP locus and had the marker recycled, leaving as integration "scar" a pUC18 plasmid backbone upstream of TRAP. The hDHFR resistance cassette was then excised by cycling through the mosquito stage

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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