RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5183

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_0309500; Gene model (P.falciparum): PF3D7_0212600; Gene product: secreted protein with altered thrombospondin repeat domain (SPATR)
Phenotype	No phenotype has been described

Last modified: 25 March 2022, 16:35

*RMgm-5183

Successful modification	The gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted	Gene disruption
Number of attempts to introduce the genetic modification	≥ 5
Reference (PubMed-PMID number)	Not published (yet)
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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	P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line	676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	Costa DM, Tavares J
Name Group/Department	IBMC – Instituto de Biologia Molecular e Celular
Name Institute	Universidade do Porto
City	Porto
Country	Portugal

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0309500

Gene Model P. falciparum ortholog

PF3D7_0212600

Gene product

secreted protein with altered thrombospondin repeat domain

Gene product: Alternative name

SPATR

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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

Yes

Name of PlasmoGEM construct/vector

PbGEM-270402

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/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood-stage growth/multiplication.

Deletion of the majority of the spatr gene (PBANKA_0309500) by double cross-over homologous recombination was attempted using the NotI-linearized PbGEM-270402 vector (PlasmoGEM, Sanger). A second construct for total gene deletion was generated by cloning the 5’ (843 bp) and 3’ (292 bp) homology regions amplified by PCR using a Taq DNA polymerase with proofreading activity (Takara) and primers P9 + P12 and P13 + P14 into the pL0001 vector (BEI Resources) via the KpnI/ClaI and EcoRI/BamHI cloning sites, respectively. The linearized constructs were used to transfect synchronized schizonts with the Nucleofector® device (Amaxa).

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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