RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-241
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Last modified: 1 March 2010, 13:28
*RMgm-241| 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) |
Reference 1 (PMID number) : 19434237 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei NK65 |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | S. Günther; K. Matuschewski; S. Müller |
| Name Group/Department | Division of Infection & Immunity and Wellcome Centre for Parasitology, Faculty of Biomedical and Lif |
| Name Institute | University of Glasgow |
| City | Glasgow |
| Country | United Kingdom, Scotland |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1413100 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1314600 | ||||||||||||||||||||||||
| Gene product | lipoate-protein ligase 1 | ||||||||||||||||||||||||
| Gene product: Alternative name | lipoic acid protein ligase A1 | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | Plasmid double cross-over | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||
| Restriction sites to linearize plasmid | KpnI/SacII | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | Gene disruptions in P. falciparum consistently were unsuccessful while in P. berghei the LplA1 gene locus was targeted by knock-in and knockout constructs. However, a LplA1(−) mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth in vitro and in vivo. In one out of six transfection experiments it was possible to amplify the diagnostic product for the PbLplA1 gene replacement after two rounds of PCR. This was verified by subcloning the PCR fragment and analysing its nucleotide sequence. However, this mutant parasite line was lost after transfer into a new animal - a procedure that is routinely performed to propagate the knockout parasite population and to be able to generate clones. These data support that the LplA1 locus can be targeted as was also shown using a control plasmid. However, the results suggest that the knockout of the LplA1 gene might have severe effects on parasite growth rate and survival during erythrocytic development. In addition to the double homologous recombination approach, an attempt was made to disrupt the lpla1 gene by single homologous recombination: The b3D.DT∧H.∧D-based knockout construct for single homologous recombination in P. berghei encompasses a 1014 bp PbLplA1 fragment corresponding to nucleotides 88 to 1098 (lacking the last 147 bp) which was amplified using the oligonucleotides Pb-3 and Pb-4 (BamHI 5′-GCGCGGATCCCAAAATATTTACTTTAATTTATCGTTGG-3′; SacII 5′-GCGCCCGCGGTTAGTCTAATGCATCTGAAAAAACATTTCC-3′). An artificial stop codon was introduced at the 3′ end of the PCR product. Transfection should result in the disruption of the gene locus and the formation of two incomplete and inactive copies of LplA1. The plasmid was digested with HpaI to linearise the vector before transfection into NK65 wild-type parasites. Three unsuccessful attempts to disrupt the gene with this construct are reported. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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