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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_0715600
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| Gene Model P. falciparum ortholog |
PF3D7_0413600
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| Gene product | 26S protease regulatory subunit 6B, putative |
| Gene product: Alternative name | Rpt3; PbRpt3 |
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| Details of the genetic modification |
| Inducable system used | No |
| Additional remarks inducable system |
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| Type of plasmid/construct used | (Linear) plasmid double cross-over |
| PlasmoGEM (Sanger) construct/vector used | Yes |
| Name of PlasmoGEM construct/vector | PbGEM-022521 |
| Modified PlasmoGEM construct/vector used | No
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| Plasmid/construct map |
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| Plasmid/construct sequence |
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| Restriction sites to linearize plasmid |
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| 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 | No |
| Additional remarks genetic modification | The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication.
To further study whether the lack of PbRpt3 could affect P. berghei blood stages life cycle, we attempted to generate knock-out (KO) lines by taking advantage of the available PlasmoGEM plasmid (PbGEM-022521; 93 % deletion of the PbRpt3 gene). Transfections were performed in two different strains of P. berghei ANKA (pG230 and PbGFP) with this KOgem construct. After selection with pyrimethamine, resistant parasites were genotyped by PCR. A total of 4 independent transfections were performed, allowing the detection of parasites 7 to 9 days after transfectionon under pyrimethamine selection. In two of these transfections, the diagnosis genotyping showed either no integration of the resistance cassette at the PbRpt3 locus, or an integration only of the 5’ side. A[er two other transfections, we detected both the integration of the construct on 5’ and 3’ sides, and the presence of the wild-type PbRpt3 gene. In order to increase the ratio of transgenic vs wild-type parasites, we first performed up to 7 successive passages of the parasites in mice under pyrimethamine regimen. However, this did not lead to any enrichment in transgenic parasites. Next, a total of five aNempts were performed to clone the parasites by limiting dilutoon either from the newly resistant parasites (first appearance a[er transfection and drug selection) or from different passages under pyrimethamine pression. Depending on the quantity of parasites injected per mouse, we either obtained no parasites in the recipient mice (3 experiments with a total of 26 recipient mice), or parasites showing both wild-type and KO construct-integrated genotype (for 1 experiment, 2/10 mice have shown parasites). In the fifth cloning experiment (20 parasites/mouse, 10 recipient mice), 2/10 mice showed blood parasites, and after passage on 3 mice, the parasites were genotyped as pure wildtype parasites. In summary, all attempts to select or clone parasites which have integrated the dhfr resistance cassette at the PbRpt3 locus were either unsuccessful or only allowed to clone wild-type parasites. These results are supportive of the essentiality of PbRpt3 during the erythrocytic stage of P. berghei. This could be interpreted as differing from the results previously obtained by Bushell et al showing a slow growing rate of PbRpt3-KO parasites at erythrocytic stages |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences  Primer information: Primers used for amplification of the target sequences 
| 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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*RMgm-5368