RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-822
|
||||||||||
Last modified: 9 February 2015, 12:45
*RMgm-822| 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 | 2 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 24498355 |
| top of page | |
| 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 507cl1 (RMgm-7) |
| Other information parent line | P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
| top of page | |
| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | J. Lin; C.J. Janse; Langsley, G |
| Name Group/Department | Leiden Malaria Research Group |
| Name Institute | Leiden University Medical Center |
| City | Leiden |
| Country | The Netherlands |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1409100 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1310600 | ||||||||||||||||||||||||
| Gene product | secretory complex protein 61 alpha | ras-related protein Rab-5B | ||||||||||||||||||||||||
| Gene product: Alternative name | Sec61-alpha; Rab5b; Rab GTPase 5b | ||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | (Linear) 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 | |||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The unsuccessful attempts to disrupt the rab5a gene indicate an essential role for blood stage growth/multiplication. P. falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. This family contains three Rab5 isoforms with PfRab5A (PF3D7_0211200) belonging to a restricted orthology group (OG4_36791) present only in Apicomplexa parasites known to invade erythrocytes (Plasmodia, Babesia and Theileria). However, PfRab5A has an insertion of 30 amino acids between the RabF1 and RabF2 motifs (Rab-effector binding motifs) that is not observed in Rab5A of Theileria and Babesia, suggesting that some PfRab5A effectors might be Plasmodium-specific. Putative PfRab5A-effectors might be involved in the uptake of haemoglobin, as GFP-tagged PfRab5A decorates vesicles containing haemoglobin. The PfRab5B orthology group (OG4_18709) is found more broadly; the C-terminus of PfRab5B (PF3D7_1310600) has no lipid-modification motif, like human Rab8 and Rab23 and ARA6. The PfRab5C (PF3D7_0106800) orthology group (OG4_10168) is the largest. PfRab5C looks like a classical Rab5, but comparison of a 3D-model of PfRab5C with the known 3D-structure of mouse Rab5C showed that these two Rab5s present different amino acids at their effector-interaction surfaces, implying that PfRab5C has the potential to recruit parasite-specific effectors. PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. Evidence is presented for a localisation at the parasite's plasma and food vacuole membranes. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s In order to create P. berghei rab5b (PBANKA_140910) gene deletion mutants, a DNA construct pL1709 was generated that would target Pbrab5b by double crossover homologous recombination. The targeting plasmid was made to target both the 5′ and 3′ regions of Pbrab5b gene and contains the drug selectable marker human dhfr (hdhfr). Prior to transfection, the plasmid was linearized by digestion with enzymes HindIII and EcoRI. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
|
Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
| |||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||