RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5341

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_0925100; Gene model (P.falciparum): PF3D7_1123200; Gene product: leucine-rich repeat protein (FBXL2 (F-box domain, leucine-rich), LRR11)
Phenotype	No phenotype has been described

Last modified: 21 April 2023, 16:42

*RMgm-5341

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	4
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 36898988
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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 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	Rashpa R, Brochet M
Name Group/Department	Faculty of Medicine, Department of Microbiology and Molecular Medicine
Name Institute	University of Geneva
City	Geneva
Country	Switzerland

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0925100

Gene Model P. falciparum ortholog

PF3D7_1123200

Gene product

leucine-rich repeat protein

Gene product: Alternative name

FBXL2 (F-box domain, leucine-rich), LRR11

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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

Modified PlasmoGEM construct/vector used

Yes
See "Additional remarks genetic modification"

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

3xHA, KO (disruption) or AID/HA targeting vectors were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of knockout and tagging constructs were performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R and goi KO-F x goi KO-R for 3xHA, AID/HA tagging and KO targeting vectors, respectively. Insertion of the GWcassette following the gateway reaction was confirmed using primer pairs GW1 x goiQCR1 and GW2 x goiQCR2.The modified library inserts were then released from the plasmid backbone using NotI. The AID/HA targeting vectors were transfected into the 615 parasite line, while the KO/GD and triple HA targeting vectors were transfected into the 2.34 line unless otherwise specified.

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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